Methods of predicting predisposition to or risk of kidney disease

ABSTRACT

Methods are disclosed herein for detecting a genetic predisposition to focal segmental glomerulosclerosis (FSGS) or hypertensive end-stage kidney disease (ESKD) or both in a human subject. The methods include detecting the presence of at least one single nucleotide polymorphism (SNP) in an APOL1 gene, such as the C-terminal exon of an APOL1 gene. In a further embodiment, methods are disclosed for detecting resistance of a subject to a disease associated with Trypanosoma infection. The methods include detecting the presence of at least one single nucleotide polymorphism (SNP) in an APOL1 gene, such as the C-terminal exon of an APOL1 gene. Also disclosed are methods for treating a subject infected with T brucei (such as T. brucei brucei, T b. rhodesiense, or T b. gambiense). The methods include administering a therapeutically effective amount of an APOL1 protein including a S342G substitution, an I384M substitution, and/or a deletion of N388 and Y389 to the subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 15/822,994, filed Nov. 27, 2017, which is adivisional application of, and claims priority to, U.S. patentapplication Ser. No. 13/642,054, filed Dec. 12, 2012 (issued as U.S.Pat. No. 9,828,637 on Nov. 28, 2017), which is a U.S. National StageApplication under 35 U.S.C. § 371 of International Application No.PCT/US2011/032924, filed Apr. 18, 2011, which claims the benefit of U.S.Provisional Application No. 61/325,343, filed Apr. 18, 2010, thedisclosure of each of which is incorporated herein by reference in itsentirety.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled 9151-228TSDV_ST25.txt, 7,436 bytes in size, generated onNov. 27, 2017 and filed via EFS-Web, is provided in lieu of a papercopy. This Sequence Listing is incorporated by reference into thespecification for its disclosures.

FIELD OF THE INVENTION

This disclosure relates to the field of individualized medicine,particularly to the determination of risk of a subject to develop renaldisease, such as focal segmental glomerulosclerosis (FSGS) or end-stagekidney disease (ESKD). The disclosure also relates to the determinationof resistance to disease associated with Trypanosoma infection andmethods for treating Trypanosoma infection in a subject.

BACKGROUND OF THE INVENTION

The prevalence of chronic kidney disease (CKD) in the United States isnow estimated at 13%, and is associated with significant morbidity andmortality (Coresh et al., Am J Kidney Dis 2003; 41(1):1-12). Inparticular, approximately 100,000 Americans develop end-stage kidneydisease (ESKD) each year. The cumulative life-time risk for ESKD variesby race, and is approximately 7.5% for African-Americans and 2.1% forEuropean Americans (Kiberd et al., J Am Soc Nephrol 2002;13(6):1635-44). In particular, African-Americans have a disproportionaterisk for several forms of CKD, among them diabetic nephropathy (Cowie etal., N Engl J Med 1989; 321(16):1074-9), hypertensive nephrosclerosis(Toto, Kidney Int Suppl 2004(92):S102-4), lupus nephritis (Fernandez etal., Arthritis Rheum 2007; 57(4):576-84), focal segmentalglomerulosclerosis (Kitiyakara et al., Am J Kidney Dis 2004;44(5):815-25) (FSGS), and HIV-associated nephropathy (a distinct form ofFSGS, also termed collapsing glomerulopathy).

FSGS is a clinical syndrome involving podocyte injury and glomerularscarring, and includes genetic forms with Mendelian inheritance,reactive forms associated with other illnesses (including HIV-1 disease)or medications, and an idiopathic form, which accounts for the majorityof cases (Barisoni et al., Clin J Am Soc Nephrol 2007; 2(3):529-42).African-Americans have a 4-fold increased risk for sporadic FSGS(Kitiyakara et al., Semin Nephrol 2003; 23(2):172-82) and an 18-fold to50-fold increased risk for HIV-1-associated FSGS (Kopp et al., KidneyInt Suppl 2003(83):S43-9; Eggers et al., J Am Soc Nephrol 2004;15(9):2477-85). Individuals of African ancestry also have increased riskfor FSGS in other geographic regions, suggesting that genetic factorscontribute to these disparities (Kitiyakara et al., Semin Nephrol 2003;23(2):172-82).

SUMMARY OF THE INVENTION

A first aspect of the invention features methods for detecting a geneticpredisposition to, or an increased risk of, the development of a renaldisease, such as focal segmental glomerulosclerosis (FSGS) orhypertensive end-stage kidney disease (ESKD), or both in a humansubject. In one embodiment, the human subject is of African (e.g., anAfrican American) or Hispanic ancestry (in preferred embodiments, thesubject of Hispanic ancestry also is of African ancestry). In anotherembodiment, the human subject is of European ancestry. The methodsinclude detecting the presence of at least one APOL1 gene risk allele(e.g., 2, 3 or 4 risk alleles; e.g., the risk allele is at least onesingle nucleotide polymorphism (SNP) in an APOL1 gene, such as theC-terminal exon of an APOL1 gene, or an inversion in an APOL1 gene(e.g., an inversion in a 5′ region of an APOL1 gene, e.g., an inversionin which the 5′ region of an APOL1 gene is replaced with a 5′ region ofan APOL4 gene). In other embodiments, the risk allele is a G1, G2, del6,and/or a G3 allele. The presence of the at least one SNP and/or the atleast one inversion determines the genetic predisposition to renaldisease, such as focal segmental glomerulosclerosis or hypertensiveESKD, or both. In other embodiments, the inversion in the APOL1 genereplaces all or a portion of up to three exons in APOL1 by sequence fromAPOL4 (e.g., the inversion may result in replacement of all or a portionof only the first exon, and/or all or a portion of the first and/orsecond exon, and/or all or a portion of the first, second, and/or all ora portion of the third exon of the APOL1 gene). These three exons maycover a range of 2000-2500 base pairs of genomic DNA (e.g., in a rangeof from about 100 base pairs to about 3000 base pairs of genomic DNA,such as a range from 1000 base pairs to about 2500 base pairs of genomicDNA), and may encode a maximum of about 420 base pairs of transcript(e.g., a range of from about 20 base pairs to about 500 base pairs oftranscript, such as from about 100 base pairs to about 420 base pairs oftranscript DNA). The actual coding sequence replaced in the APOL1protein may only code for about 1 to about 30 amino acids, e.g., about10 to about 20 amino acids, e.g., about 14 amino acids from APOL4. Thesubstituted amino acids in the APOL1 protein may all appear in thepreprotein portion of the hybrid APOL4/APOL1 protein and all or aportion of the replaced amino acids may be cleaved depending upon theextent and actual sequence of the inversion. In an embodiment, theinversion occurs in a coding and/or non-coding region of the APOL1 geneand/or results in a functional gene product.

In other embodiments, the method includes taking a sample from the humansubject to be tested. In still other embodiments, the at least one SNPis a G at rs73885319; a G at rs60910145; a 6 base pair deletion atrs71785313; and/or a combination thereof. The at least one SNP mayproduce an APOL1 polypeptide having a serine to glycine mutation atposition 342 (S342G), an isoleucine to methionine mutation at position384 (I384M), a deletion of amino acids N388 and Y389, and/or acombination thereof (e.g., the at least one SNP produces an APOL1polypeptide having a S342G and an I384M mutation). In yet otherembodiments, the method includes determining the presence of the atleast one SNP and/or the at least one inversion on both chromosomes ofthe subject. In another embodiment, the subject is infected with humanimmunodeficiency virus (HIV) and is at a greater risk of developingFSGS. In still other embodiments, the subject is homozygous orheterozygous for the at least one SNP and/or the inversion. In anembodiment, a determination that the human subject is homozygous for theat least one SNP and/or the at least one inversion indicates anincreased likelihood the human subject will develop renal diseaserelative to a human subject that is heterozygous for the at least oneSNP and/or the at least one inversion.

In another embodiment, the presence of the at least one SNP and/or theat least one inversion indicates the human subject has an increased riskof renal disease following treatment with a therapeutic. For example, asubject having one or more APOL1 gene risk alleles may need to beoffered a treatment regimen with respect to blood pressure medications,steroids, and/or immunosuppressive agents that is different from asubject lacking any (or only having, e.g., one)APOL1 gene risk allele.In particular, subjects having one or more APOL1 gene risk alleles aremore susceptible to renal damage and/or disease and the risk of kidneydamage increases in patients having one or more APOL1 gene risk allelesthat are treated with blood pressure medications, steroids, and/orimmunosuppressive agents. Thus, in patients having one or more APOL1gene risk alleles, the concentration of a given blood pressuremedication, steroid, and/or immunosuppressive agent and/or the length oftreatment may need to be decreased relative to a patient lacking any (orhaving only one) APOL1 gene risk alleles. Examples of therapeuticsinclude blood pressure medications (e.g., a diuretic (e.g.,chlorthalidone, chlorothiazide, furosemide, hydrochlorothiazide,indapamide, metolazone, amiloride hydrochloride, spironolactone,triamterene, bumetanide, or a combination thereof), an alpha adrenergicantagonist (e.g., alfuzosin, doxazosin, prazosin, terazosin, ortamsulosin, or a combination thereof), a central adrenergic inhibitor(e.g., clonidine, guanfacine, or methyldopa, or a combination thereof),an angiotensin converting enzyme (ACE) inhibitor (e.g., benazepril,captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril,quinapril, ramipril, or trandolapril, or combinations thereof), anangiotensin II receptor blocker (e.g., candesartan, eprosartan,irbesartan, losartan, olmesartan, telmisartan, or valsartan, orcombinations thereof), an alpha blocker (e.g., doxazosin, prazosin, orterazosin, or a combination thereof), a beta blocker (e.g., acebutolol,atenolol, betaxolol, bisoprolol, carteolol, metoprolol, nadolol,nebivolol, penbutolol, pindolol, propranolol, solotol, or timolol, or acombination thereof), a calcium channel blocker (e.g., amlodipine,bepridil, diltiazem, felodipine, isradipine, nicardipine, nifedipine,nisoldipine, or verapamil, or combination thereof), a vasodilator (e.g.,hydralazine or minoxidil, or combination thereof), and a renin inhibitor(e.g., aliskiren), or combinations thereof), a steroid (e.g., acorticosteroid, such as cortisone, prednisone, methylprednisolone, orprednisolone), or an anabolic steroid (anatrofin, anaxvar, annadrol,bolasterone, decadiabolin, decadurabolin, dehydropiandrosterone (DHEA),delatestryl, dianiabol, dihydrolone, durabolin, dymethazine,enoltestovis, equipose, gamma hydroxybutyrate, maxibolin, methatriol,methyltestosterone, parabolin, primobolin, quinolone, therabolin,trophobolene, and winstrol), or an immunosuppressive agent, such as aglucocorticoid, a cytostatic, an antibody, or an anti-immunophilinand/or mychophenolate mofetil (MMF), FK-506, azathioprine,cyclophosphamide, methotrexate, dactinomycin, antithymocyte globulin(ATGAM), an anti-CD20-antibody, a muromonoab-CD3 antibody, basilizimab,daclizumab, cyclosporin, tacrolimus, voclosporin, sirolimus, aninterferon, infliximab, etanercept, adalimumab, fingolimod, and/ormyriocin).

In other embodiments, the presence of the at least one SNP and/or the atleast one inversion indicates the human subject has an increased risk ofkidney failure (and may have a greater need for a kidney transplant)relative to a human subject lacking the at least one SNP and/or the atleast one inversion.

A second aspect of the invention features a method of evaluating a humansubject (e.g., a potential kidney donor) for their suitability as atransplant donor by determining the presence of at least one human APOL1gene risk allele (e.g., at least one (e.g., two, three, or four) singlenucleotide polymorphism (SNP; e.g., a G at rs73885319 (G1), a G atrs60910145 (G2), a 6 base pair deletion at rs71785313 (del6), and/or acombination thereof) and/or at least one inversion in a human APOL1gene) in a cell, tissue, or organ of the human subject, in which thepresence of the at least one APOL1 gene risk allele indicates the humansubject is not suitable as a transplant donor. In other embodiments, theat least one SNP produces an APOL1 polypeptide having a serine toglycine mutation at position 342 (S342G), an isoleucine to methioninemutation at position 384 (I384M), a deletion of amino acids N388 andY389, and/or a combination thereof (e.g., the at least one SNP producesan APOL1 polypeptide having a S342G and an I384M mutation). In otherembodiments, the inversion is, e.g., a substitution of the 5′ region ofthe APOL1 gene with the 5′ region of another apolipoprotein gene (e.g.,an APOL4 gene)). In yet other embodiments, the method includesdetermining the presence of the at least one SNP and/or the at least oneinversion on both chromosomes of the subject. The human subject may beof African or Hispanic ancestry (e.g., an African American subject). Inother embodiments, the method includes detecting the presence of the atleast one SNP and the at least one inversion in said APOL1 gene; theinversion includes recombination between the human APOL1 gene andanother apolipoprotein gene (e.g., a human APOL4 gene); the inversionoccurs in a coding and/or non-coding region of the APOL1 gene; and/orthe inversion results in a functional gene product. In still otherembodiments, detection of at least two SNPs in the human subject furtherindicates the human subject is not suitable as a transplant donor. Themethod may further include determining whether the human subject ishomozygous or heterozygous for the at least one SNP and/or the at leastone inversion (e.g., a determination that the human subject ishomozygous for the at least one SNP and/or the at least one inversionindicates an increased likelihood the human subject will develop renaldisease relative to a human subject that is heterozygous for the atleast one SNP and/or the at least one inversion).

A third aspect of the invention features methods for detecting a diseaseassociated with Trypanosoma spp. infection, such as a disease associatedwith T. brucei infection, such as African trypanosomiasis (sleepingsickness) in a subject (e.g., a human subject) by detecting a resistanceallele of an APOL1 gene. In an embodiment, the resistance alleleincludes at least one (e.g., two, three, or four) SNP in an APOL1 gene,such as the C-terminal exon of an APOL1 gene, and/or at least oneinversion in an APOL1 gene (e.g., an inversion in a 5′ region of anAPOL1 gene, e.g., an inversion in which the 5′ region of an APOL1 geneis replaced with a 5′ region of an APOL4 gene). In other embodiments,the resistance allele is a G1, G2, del6, and/or a G3 allele. Thepresence of the SNP and/or the inversion determines resistance of thesubject to disease associated with Trypanosoma spp. infection.

A fourth aspect of the invention features methods for treating a subjectinfected with T. brucei (such as T. brucei brucei, T b. rhodesiense, orT b. gambiense). The methods include administering a therapeuticallyeffective amount of an APOL1 protein (e.g., a human APOL1 protein) thatincludes at least one resistance allele (e.g., a S342G substitution, anI384M substitution, a deletion of N388 and Y389 and/or an inversion tothe subject. In some examples, the APOL1 protein is included in humanserum administered to the subject. In other examples, the APOL1 proteinis recombinant.

The foregoing and other features of the disclosure will become moreapparent from the following detailed description, which proceeds withreference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show a pair of plots showing logistic regressionadjusting for APOL1 alleles G1 and G2. Results of association between205 idiopathic biopsy-proven African-American FSGS cases and 180African-American controls. On the x-axis and y-axis, genomic positionand −log 10 of the p-values are shown. Highlighted are also SNPsrs4821481 and rs3752462 whose combined risk alleles define the E-1haplotype (Kopp et al., Nature Genet. 40:1175-1184, 2008). (1A)Association of the studied variants with FSGS using Fisher's exact test.(1B) Association of the studied variants with FSGS after adjusting forallele G1 using logistic regression.

FIG. 2 is a graph showing extended haplotype homozygosity (EHH) valuesfor the three APOL1 alleles computed after combining Hapmap phase 2genotype data with genotype data for alleles G1 and G2 for Yorubasamples. This suggests an older age for allele G2, although largeuncertainty is introduced by the fact that only 8 haplotypes with the G2allele were found in the Yoruba sample set.

FIG. 3 shows a pair of maps showing average annual rate of new cases of(Panel A) Trypanosoma brucei rhodesiense and (Panel B) Trypanosomabrucei gambiense sleeping sickness reported between 1997 and 2006 inAfrica.

FIG. 4 shows a series of panels showing trypanolytic potential of ApoL1variants on NETS-resistant (SRA+) and NETS-sensitive (SRA−) T b.rhodesiense ETat 1.2 (Edinburgh Trypanozoon antigenic type 1, clone 2)clones. (Panel A) Titration of trypanolytic activity in plasma samplesafter overnight incubation (ctrl=control incubation in fetal calf serumwithout plasma; hom, het=homozygous and heterozygous mutations,respectively). (Panel B) ApoL1 content of various plasma samples beforeand after affinity chromatography through SRA column (NHS=normal humanserum; WT=wild type apoL1; S=serine 342; G=glycine 342; I=isoleucine384; M=methionine 384; i=insertion of N388/Y389; d=deletion ofN388/Y389). (Panel C) Trypanolytic activity of various recombinant ApoL1variants after overnight incubation (FCS=fetal calf serum). (Panel D)Kinetics of trypanolysis by 20 μg/ml recombinant ApoL1 variants in thepresence or absence of 25 μM chloroquine (clq). (Panel E) Phenotype ofETat1.2R trypanosomes incubated with various recombinant ApoL1 (20μg/ml; 1 h30 and 6 h incubation, for G1 and G2 respectively; the arrowspoint to the swelling lysosome).

FIG. 5 shows the mean age at dialysis initiation for subjects by APOL1risk allele status. Due to the proximity of the alleles it is notexpected for a diploid sample to have more then two risk alleles, onlysix groups existed within this dataset; Wt+Wt, Wt+G1, G1+G1, Wt+G2,G2+G2, G1+G2. Bar height is the mean age. Error bars denote the standarderror.

*significantly different from wild type (Wt+Wt)

FIG. 6 shows the mean age at dialysis for subjects with a G1 riskallele. Panel A shows the mean age at dialysis initiation by G1 riskallele status in subjects with hypertension attributed ESRD (H-ESKD).Panel B shows age by G1 risk allele status in subjects with other ESRDcauses, including HIV, inflammation, toxins, etc. Horizontal bars denotemean age while vertical bars denote standard error.

*significantly different from wild type (Wt+Wt)

FIG. 7. Panel A is a schematic showing the relationship of the APOL1,APOL2, and APOL4 genes on chromosome 22. The G1 and G2 alleles are alsoshown. Panel B is a schematic showing the inversion of a segment of DNAincluding the 5′ end of APOL4, all of APOL2, and the 5′ end of APOL1.

FIG. 8 is a graph of HapMap gene expression data that showing thecoordinated regulation of APOL1 and APOL2.

FIG. 9 is a photograph of a gel showing the presence of a APOL1-APOL4hybrid gene following PCR amplification from 12 human samples. Lane 1shows a size ladder. Lanes 4 and 6 show the inversion.

FIG. 10 shows the genomic sequence of APOL1 and APOL4 following G3inversion (SEQ ID NO: 7).

FIG. 11. Panel A shows a potential transcript formed in individuals withthe G3 inversion and another SNP, rs9610445 (the C allele), in which anessential splice site is eliminated. The donor splice site sequence ofAPOL4 (SEQ ID NO: 8) and the acceptor splice site of APOL1 (SEQ ID NO:9) are shown. Panel B is a potential transcript formed in individualswith the G3 inversion and another SNP, rs6000181 T (minor) allele, inwhich a methionine start site is eliminated.

SEQUENCE LISTING

The nucleic acid sequences and amino acid sequences listed are shownusing standard letter abbreviations for nucleotide bases, and threeletter code for amino acids, as defined in 37 C.F.R. 1.822. Only onestrand of each nucleic acid sequence is shown, but the complementarystrand is understood as included by any reference to the displayedstrand. It should be noted that single nucleotide polypmorphisms areidentified in the leading strand, wherein the risk nucleotide is listedfirst, and the protective nucleotide is listed second. Due to thecomplementary nature of DNA, the single nucleotide polymorphism ispresent in both DNA strands, and thus can also be identified in thelagging strand.

SEQ ID NOs: 1-3 are nucleic acid sequences from the APOL1 gene, eachinclude a single nucleotide polymorphism of interest.

SEQ ID NOs: 4 and 5 are exemplary nucleic acid and amino acid sequencesof a human apolipoprotein L1, respectively.

SEQ ID NO: 7 is a genomic sequence of an APOL1 and APOL4 inversion.

DETAILED DESCRIPTION I. Abbreviations and Terms

APOL1: apolipoprotein L1 gene or protein

ESKD: end-stage kidney disease

FSGS: focal segmental glomerulosclerosis

HIV: human immunodeficiency virus

LD: linkage disequilibrium

LOD: logarithm of the odds

MALD: mapping by admixture linkage disequilibrium

NHS: normal human serum

OR: odds ratio

ROC: receiver operator characteristic

SNP: single nucleotide polymorphism

SRA: serum resistance-associated gene or protein

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

Definitions of common terms in molecular biology may be found inBenjamin Lewin, Genes V, published by Oxford University Press, 1994(ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia ofMolecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

Administration: To provide or give a subject an agent by any effectiveroute. Exemplary routes of administration include, but are not limitedto, oral, injection (such as subcutaneous, intramuscular, intradermal,intraperitoneal, and intravenous), sublingual, rectal, transdermal,intranasal, vaginal and inhalation routes. Administration ofextracorporeal treatment (e.g., dialysis) is also included.

African ancestry: An individual whose ancestors are from Sub-SaharanAfrica prior to the era of European expansion (prior to about 1500).There are a number of programs that can be used to analyze DNA todetermine if an individual is of African ancestry, such as STRUCTURE™(available on the internet at pritch.bsd.uchicago.edu/structure.html).In one example, African-American individuals are those individuals whoreside in the United States and self-identify themselves as being ofAfrican origin. In another example, African-Americans are individualswho reside in the United States and self-identify as being of Africanorigin, and are of African ancestry as determined by a program thatanalyzes DNA ancestry, such as STRUCTURE™.

Allele: A particular form of a genetic locus, distinguished from otherforms by its particular nucleotide sequence, or one of the alternativepolymorphisms found at a polymorphic site.

Allele frequency: A measure of the relative frequency of an allele at agenetic locus in a population. Usually allele frequency is expressed asa proportion or a percentage. In population genetics, allele frequenciesare used to depict the amount of genetic diversity at the individual,population, or species level. There are various databases in the publicdomain that contain SNPs and a user may for example, determine therelative allele frequency in some instances using such publiclyavailable databases.

In the instant application the allele frequency for a risk allele isgreater than 5% in subjects of African ancestry. In a furtherembodiment, the allele frequency for a risk allele is greater than atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, or at least 50% in subjects ofAfrican ancestry. In some embodiments, an unaffected population is usedto calculate allele frequency. Risk is elevated in individuals thatcarry the risk allele.

There are a number of diseases or disorders that are associated with theidentification of one or more SNPs or risk alleles. In many cases, thediseases or disorders are autosomal dominant mutations and anyassociated SNPs are observed only in individuals who present withclinical manifestations of the disease. In other circumstances, theoccurrence of a disease-associated SNP is so rare that no-knownfrequency can be determined (for example, through the use of publicdomain SNP databases or by comparison with the literature) and thesediseases/disorders are correctly defined as having an allele frequencysignificantly lower than 1%.

Amplification: To increase the number of copies of a nucleic acidmolecule. The resulting amplification products are called “amplicons.”Amplification of a nucleic acid molecule (such as a DNA or RNA molecule)refers to use of a technique that increases the number of copies of anucleic acid molecule in a sample. An example of amplification is thepolymerase chain reaction (PCR), in which a sample is contacted with apair of oligonucleotide primers under conditions that allow for thehybridization of the primers to a nucleic acid template in the sample.The primers are extended under suitable conditions, dissociated from thetemplate, re-annealed, extended, and dissociated to amplify the numberof copies of the nucleic acid. This cycle can be repeated. The productof amplification can be characterized by such techniques aselectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing.

Other examples of in vitro amplification techniques include quantitativereal-time PCR; reverse transcriptase PCR (RT-PCR); real-time PCR (rtPCR); real-time reverse transcriptase PCR (rt RT-PCR); nested PCR;strand displacement amplification (see U.S. Pat. No. 5,744,311);transcription-free isothermal amplification (see U.S. Pat. No.6,033,881); repair chain reaction amplification (see PCT Publication No.WO 90/01069); ligase chain reaction amplification (see European patentpublication No. EP-A-320 308); gap filling ligase chain reactionamplification (see U.S. Pat. No. 5,427,930); coupled ligase detectionand PCR (see U.S. Pat. No. 6,027,889); and NASBA™ RNA transcription-freeamplification (see U.S. Pat. No. 6,025,134), amongst others.

APOL1: A gene encoding human apolipoprotein L, 1 (OMIM: 603743). Thisgene encodes a secreted high density lipoprotein which binds toapolipoprotein A-I. Apolipoprotein A-I is a relatively abundant plasmaprotein and is the major apoprotein of HDL. Several different transcriptvariants encoding different isoforms have been found for this gene.

Exemplary SNPs observed in APOL1 that are associated with apredisposition to renal disease include a G at rs73885319 (“G1”); a G atrs60910145 (“G2”); or a 6 base pair deletion at rs71785313 (“del6”)(incorporated by reference as present in dbSNP (ncbi.nlm.nih.gov/SNP) onApr. 18, 2010), as well as combinations thereof.

Nucleic acid and protein sequences for human APOL1 are publiclyavailable. For example, GENBANK® Accession No. NC_000022.10 (nucleotides36649117 . . . 36663577) discloses an exemplary human APOL1 genomicsequence (incorporated by reference as provided by GENBANK® on Apr. 18,2010). In other examples, GENBANK® Accession Nos. AF305224.1,NM_003661.3, NM_145343.2, NM_001136540.1, z82215, and BC127186.1disclose exemplary human APOL1 nucleic acid sequences, and GENBANK®Accession Nos. CAQ09089, NP_003652, AAI43039.1, and AAI42721.1 discloseexemplary human APOL1 protein sequences, all of which are incorporatedby reference as provided by GENBANK® on Apr. 18, 2010.

Caucasian: A human racial classification traditionally distinguished byphysical characteristics such as very light to brown skin pigmentationand straight to wavy or curly hair, which includes persons havingorigins in any of the original peoples of Europe, North Africa, or theMiddle East. Popularly, the word “white” is used synonymously with“Caucasian” in North America. Such persons retain substantial geneticsimilarity to natives or inhabitants of Europe, North Africa, or theMiddle East. In a particular example, a Caucasian is at least 1/64Caucasian.

Concordance: The presence of two or more loci or traits (or combinationthereof) derived from the same parental chromosome. The opposite ofconcordance is discordance, that is, the inheritance of only one (of twoor more) parental alleles and/or traits associated with a parentalchromosome.

Correlation: A correlation between a phenotypic trait and the presenceor absence of a genetic marker (or haplotype or genotype) can beobserved by measuring the phenotypic trait and comparing it to datashowing the presence or absence of one or more genetic markers. Somecorrelations are stronger than others, meaning that in some instancessubjects with FSGS will display a particular genetic marker (e.g., 100%correlation). In other examples the correlation will not be as strong,meaning that a subject with FSGS will only display a particular geneticmarker 90%, 85%, 70%, 60%, 55%, or 50% of the time. In some instances, ahaplotype which contains information relating to the presence or absenceof multiple markers can also be correlated to a genetic predispositionsuch as the development of FSGS, or the type of onset. Correlations canbe described using various statistical analyses known to the skilledartisan.

Decrease: Becoming less or smaller, as in number, amount, size, orintensity. In one example, decreasing the risk of a disease (such asFSGS or hypertensive ESKD) includes a decrease in the likelihood ofdeveloping the disease by at least about 20%, for example by at leastabout 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In another example,decreasing the risk of a disease includes a delay in the development ofthe disease, for example a delay of at least about six months, such asabout one year, such as about two years, about five years, or about tenyears.

In one example, decreasing the signs and symptoms of FSGS includesdecreasing the effects of the disease such as podocyte injury orglomerular scarring by a desired amount, for example by at least 5%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 50%, at least 75%, or even at least 90%, as compared to a responsein the absence of a therapeutic composition.

In another, decreasing the signs and symptoms of Trypanosoma infection,such as sleeping sickness, includes decreasing the effects of thedisease such as fever, headache, joint pain, lymph node swelling,anemia, confusion, reduced coordination, or disruption of the sleepcycle by a desired amount, for example by at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 50%, atleast 75%, or even at least 90%, as compared to a response in theabsence of a therapeutic composition.

DNA (deoxyribonucleic acid): DNA is a long chain polymer which comprisesthe genetic material of most living organisms (some viruses have genescomprising ribonucleic acid (RNA)). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine, guanine, cytosine and thymine bound to a deoxyribosesugar to which a phosphate group is attached. Triplets of nucleotides(referred to as codons) code for each amino acid in a polypeptide, orfor a stop signal (termination codon). The term codon is also used forthe corresponding (and complementary) sequences of three nucleotides inthe mRNA into which the DNA sequence is transcribed.

Unless otherwise specified, any reference to a DNA molecule is intendedto include the reverse complement of that DNA molecule. Except wheresingle-strandedness is required by the text herein, DNA molecules,though written to depict only a single strand, encompass both strands ofa double-stranded DNA molecule. Thus, a reference to the nucleic acidmolecule that encodes a protein, or a fragment thereof, encompasses boththe sense strand and its reverse complement. Thus, for instance, it isappropriate to generate probes or primers from the reverse complementsequence of the disclosed nucleic acid molecules.

Dominant Model: A genetic based model that tests the association ofhaving at least one risk allele (e.g. Dd or DD) versus not having a riskallele at all (dd).

End-stage kidney disease (ESKD) or end-stage renal disease (ESRD): Astage of kidney impairment that is irreversible and cannot be controlledby conservative management alone. ESKD requires dialysis or kidneytransplantation to maintain life.

European ancestry: A type of ancestry shared by people who derived fromthe fertile crescent of the Middle East some 50,000 years ago and spreadto occupy Europe, the Middle East, parts of Eurasia and South Asia.There are a number of programs that can be used to analyze DNA todetermine if an individual is of African ancestry, such as STRUCTURE™(available on the internet at pritch.bsd.uchicago.edu/structure.html)and EURODNA™ and ANCESTRYBYDNA™ (available through the DNA printwebsite). In one example, European-American individuals are thoseindividuals who reside in the United States and self-identify themselvesas being of European origin. In another example, European-Americans areindividuals who reside in the United States and self-identify as beingof European origin, and are of European ancestry as determined by aprogram that analyzes DNA ancestry.

Focal segmental glomerulosclerosis (FSGS): A clinical syndrome involvingpodocyte injury and glomerular scarring, and includes genetic forms withMendelian inheritance, reactive forms associated with other illnesses(including HIV-1 disease) or medications, and an idiopathic form, whichaccounts for the majority of cases. The name refers to the appearance ofthe kidney tissue on biopsy: focal—only some of the glomeruli areinvolved; segmental—only part of an entire glomerulus is involved;glomerulosclerosis—scarring of the glomerulus. FSGS presents as anephrotic syndrome (which is characterized by edema (associated withweight gain), hypoalbuminemia (low serum albumin (a protein) in theblood), hyperlipidemia and hypertension (high blood pressure)). Inadults it may also present as kidney failure and proteinuria, without afull-blown nephrotic syndrome.

There are five mutually exclusive variants of FSGS that can bedistinguished by the pathologic findings seen on renal biopsy:collapsing variant, glomerular tip lesion variant, cellular variant,perihilar variant, and not otherwise specified (NOS) variant.Determining the type of variant can have prognostic value in individualswith primary FSGS (where no underlying cause is determined). Thecollapsing variant is associated with higher rate of progression toend-stage renal disease, whereas glomerular tip lesion variant has lowrate of progression to end-stage renal disease in most patients. Thecellular variant shows a similar clinical presentation to collapsing andglomerular tip variant but has intermediate outcomes between these twovariants.

Genetic predisposition: Susceptibility of a subject to a disease, suchas renal disease, including FSGS and hypertensive end stage renaldisease. Detecting a genetic predisposition can include, but does notnecessarily include, detecting the presence of the disease itself, suchas but not limited to an early stage of the disease process. Detecting agenetic predisposition also includes detecting the risk of developingthe disease, and determining the susceptibility of that subject todeveloping the disease or to having a poor prognosis for the disease.Thus, if a subject has a genetic predisposition to a disease they do notnecessarily develop the disease but are at risk for developing thedisease.

Genomic target sequence: A sequence of nucleotides located in aparticular region in the human genome that corresponds to one or morespecific genetic abnormalities, such as a nucleotide polymorphism, adeletion, an insertion, or amplification. The target can be for instancea coding sequence; it can also be the non-coding strand that correspondsto a coding sequence. The target can also be a non-coding sequence, suchas an intronic sequence. In some examples, genomic target sequences aregenomic sequences of genes that encode apolipoprotein L1(APOL1).

Gene: A segment of DNA that contains the coding sequence for a protein,wherein the segment may include promoters, exons, introns, and otheruntranslated regions that control expression.

Genotype: An unphased 5′ to 3′ sequence of nucleotide pair(s) found at aset of one or more polymorphic sites in a locus on a pair of homologouschromosomes in an individual. “Genotyping” is a process for determininga genotype of an individual.

Haplotype: A 5′ to 3′ sequence of nucleotides found at a set of one ormore polymorphic sites in a locus on a single chromosome from a singleindividual. “Haplotype pair” is the two haplotypes found for a locus ina single individual. With regard to a population, haplotypes are theordered, linear combination of polymorphisms (e.g., single nucleotidepolymorphisms (SNPs)) in the sequence of each form of a gene (onindividual chromosomes) that exist in the population. “Haplotyping” is aprocess for determining one or more haplotypes in an individual andincludes use of family pedigrees, molecular techniques and/orstatistical inference. “Haplotype data” is the information concerningone or more of the following for a specific gene: a listing of thehaplotype pairs in an individual or in each individual in a population;a listing of the different haplotypes in a population; frequency of eachhaplotype in that or other populations, and any known associationsbetween one or more haplotypes and a trait.

Haplotype block: Sites of closely located SNPs which are inherited inblocks. A haplotype block includes a group of SNP locations that do notappear to recombine independently and that can be grouped together.Regions corresponding to blocks have a few common haplotypes whichaccount for a large proportion of chromosomes. Identification ofhaplotype blocks is a way of examining the extent of linkagedisequilibrium (LD) in the genome. The “Hap-Map” project (see theinternet at the Hap-Map website) describes the mapping of haplotypeblocks in the human genome.

There are programs available on the internet for the identification ofhaplotype blocks, such as the program HAPBLOCK™ which runs on both PCand Unix and is available from the University of Southern Californiawebsite on the internet. A further program, which in addition to blockidentification also has visualization and selection of “tagging” SNPs isHAPLOBLOCKFINDER™, which runs interactively on the web or can bedownloaded for local machine use (Unix or PC). It can be accessed at theprogram website available on the internet.

Hispanic Ancestry: A person of Mexican, Puerto Rican, Cuban, Dominican,South or Central American, or other Spanish or Portuguese culture ororigin, regardless of race.

Hybridization: Oligonucleotides and their analogs hybridize by hydrogenbonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary bases. Generally, nucleic acidsconsist of nitrogenous bases that are either pyrimidines (cytosine (C),uracil (U), and thymine (T)) or purines (adenine (A) and guanine (G)).These nitrogenous bases form hydrogen bonds between a pyrimidine and apurine, and the bonding of the pyrimidine to the purine is referred toas “base pairing.” More specifically, A will hydrogen bond to T or U,and G will bond to C. “Complementary” refers to the base pairing thatoccurs between two distinct nucleic acid sequences or two distinctregions of the same nucleic acid sequence. For example, anoligonucleotide can be complementary to a specific genetic locus, so itspecifically hybridizes with a mutant allele (and not the referenceallele) or so that it specifically hybridizes with a reference allele(and not the mutant allele).

“Specifically hybridizable” and “specifically complementary” are termsthat indicate a sufficient degree of complementarity such that stableand specific binding occurs between the oligonucleotide (or its analog)and the DNA or RNA target. The oligonucleotide or oligonucleotide analogneed not be 100% complementary to its target sequence to be specificallyhybridizable. An oligonucleotide or analog is specifically hybridizablewhen binding of the oligonucleotide or analog to the target DNA or RNAmolecule interferes with the normal function of the target DNA or RNA,and there is a sufficient degree of complementarity to avoidnon-specific binding of the oligonucleotide or analog to non-targetsequences under conditions where specific binding is desired, forexample under physiological conditions in the case of in vivo assays orsystems. Such binding is referred to as specific hybridization. In oneexample, an oligonucleotide is specifically hybridizable to DNA or RNAnucleic acid sequences including an allele of a gene, wherein it willnot hybridize to nucleic acid sequences containing a polymorphism.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method ofchoice and the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (especially the Na⁺ concentration) of the hybridization bufferwill determine the stringency of hybridization, though wash times alsoinfluence stringency. Calculations regarding hybridization conditionsrequired for attaining particular degrees of stringency are discussed bySambrook et al. (ed.), Molecular Cloning: A Laboratory Manual, 2nd ed.,vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1989, chapters 9 and 11.

The following is an exemplary set of hybridization conditions and is notlimiting:

Very High Stringency (Detects Sequences that Share at Least 90%Identity)

Hybridization: 5×SSC at 65° C. for 16 hours

Wash twice: 2×SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5×SSC at 65° C. for 20 minutes each

High Stringency (Detects Sequences that Share at Least 80% Identity)

Hybridization: 5×-6×SSC at 65° C.-70° C. for 16-20 hours

Wash twice: 2×SSC at RT for 5-20 minutes each

Wash twice: 1×SSC at 55° C.-70° C. for 30 minutes each

Low Stringency (Detects Sequences that Share at Least 50% Identity)

Hybridization: 6×SSC at RT to 55° C. for 16-20 hours

Wash at least twice: 2×-3×SSC at RT to 55° C. for 20-30 minutes each.

Hypertension: High blood pressure; transitory or sustained elevation ofsystemic arterial blood pressure to a level likely to inducecardiovascular damage or other adverse consequences. Hypertension hasbeen arbitrarily defined as a systolic blood pressure above 140 mm Hg ora diastolic blood pressure above 90 mm Hg. Consequences of uncontrolledhypertension include retinal vascular damage (Keith-Wagener-Barkerchanges), cerebrovascular disease and stroke, left ventricularhypertrophy and failure, myocardial infarction, dissecting aneurysm, andrenovascular disease. An underlying disorder (such as renal disease,Cushing syndrome, pheochromocytoma) is identified in fewer than 10% ofall cases of hypertension. The remainder, traditionally labeled“essential” hypertension, probably arise from a variety of disturbancesin normal pressure-regulating mechanisms (which involve baroreceptors,autonomic influences on the rate and force of cardiac contraction andvascular tone, renal retention of salt and water, formation ofangiotensin II under the influence of renin and angiotensin-convertingenzyme, and other factors known and unknown), and most are probablygenetically conditioned.

Hypertensive nephropathy (or “hypertensive nephrosclerosis,” or“hypertensive renal disease” or “hypertensive kidney disease”): Amedical condition referring to damage to the kidney due to chronic highblood pressure. In the kidneys, as a result of benign arterialhypertension, hyaline (pink, amorphous, homogeneous material)accumulates in the wall of small arteries and arterioles, producing thethickening of their walls and the narrowing of the lumens—hyalinearteriolosclerosis. Consequent ischemia produces tubular atrophy,interstitial fibrosis, glomerular alterations (smaller glomeruli withdifferent degrees of hyalinization—from mild to sclerosis of glomeruli)and periglomerular fibrosis. In advanced stages (“end-stage”), renalfailure will occur.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein or organelle) has been substantially separated orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, e.g., otherchromosomal and extra-chromosomal DNA and RNA, proteins and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinantexpression in a host cell as well as chemically synthesized nucleicacids.

Linkage: The association of two or more loci at positions on the samechromosome, such that recombination between the two loci is reduced to aproportion significantly less than 50%. The term linkage can also beused in reference to the association between one or more loci and atrait if an allele (or alleles) and the trait, or absence thereof, areobserved together in significantly greater than 50% of occurrences. Alinkage group is a set of loci, in which all members are linked eitherdirectly or indirectly to all other members of the set LinkageDisequilibrium: Co-occurrence of two genetic loci (e.g., markers) at afrequency greater than expected for independent loci based on the allelefrequencies.

Linkage disequilibrium (LD) typically occurs when two loci are locatedclose together on the same chromosome. When alleles of two genetic loci(such as a marker locus and a causal locus) are in strong LD, the alleleobserved at one locus (such as a marker locus) is predictive of theallele found at the other locus (for example, a causal locuscontributing to a phenotypic trait). The linkage disequilibrium (LD)measure r² (the squared correlation coefficient) can be used to evaluatehow SNPs are related on a haplotype block. For each tag SNP, the r²between that tag SNP and each additional SNP in a genotyping set can becalculated. The highest of these values is the maximum r² value, m. Inseveral embodiments, a haplotype block can be identified by SNPs thathave an r² value of greater than or equal to 0.75, greater than or equalto 0.8, greater than or equal to about 0.85, greater than or equal to0.9, or greater than or equal to 0.95 from the tag SNP. A low r² valuesuch as less than or equal to 0.3, less than or equal to 0.2, less thanor equal to 0.1, is generally considered to be less predictive than ahigher r² value, which is considered a stronger predictor of linkagedisequilibrium, such as greater than or equal to 0.75.

Locus: A location on a chromosome or DNA molecule corresponding to agene or a physical or phenotypic feature, where physical featuresinclude polymorphic sites.

Mutation: Any change of a nucleic acid sequence as a source of geneticvariation. For example, mutations can occur within a gene or chromosome,including specific changes in non-coding regions of a chromosome, forinstance changes in or near regulatory regions of genes. Types ofmutations include, but are not limited to, base substitution pointmutations (which are either transitions or transversions), deletions,and insertions. Missense mutations are those that introduce a differentamino acid into the sequence of the encoded protein; nonsense mutationsare those that introduce a new stop codon; and silent mutations arethose that introduce the same amino acid often with a base change in thethird position of the codon. In the case of insertions or deletions,mutations can be in-frame (not changing the frame of the overallsequence) or frame shift mutations, which may result in the misreadingof a large number of codons (and often leads to abnormal termination ofthe encoded product due to the presence of a stop codon in thealternative frame).

Non-coding: A change in nucleotide sequence that does not result in theproduction of a codon that encodes for an amino acid other than thewild-type human sequence, and therefore does not result in theproduction of any alteration in polypeptide sequence. In the instantapplication, the term “non-coding” refers to the exclusion ofnon-synonymous SNPs or haplotypes. In addition, the term “non-coding”also excludes promoter regions of a gene and is therefore limited tointronic and exonic domains of the gene.

Odds Ratio: A calculation performed by analysis of a two by twocontingency table. In one example, the first column provides a riskindicator in the absence of a disease (e.g., FSGS). The second columnprovides the same risk indicator in the presence of the same disease.The first row lists the risk indicator in the absence of a risk factor(such as race) and the second row lists the same risk indicator in thepresence of the same risk factor (e.g., race). The Odds Ratio (OR) isdetermined as the product of the two diagonal entries in the contingencytable divided by the product of the two off-diagonal entries of thecontingency table. An OR of 1 is indicative of no association.Accordingly, very large or very small ORs are indicative of a strongassociation between the factors under investigation. The OR isindependent of the ratio of cases or controls in a study, group orsubset.

Oligonucleotide: An oligonucleotide is a plurality of joined nucleotidesjoined by native phosphodiester bonds, between about 6 and about 300nucleotides in length. An oligonucleotide analog refers to moieties thatfunction similarly to oligonucleotides but have non-naturally occurringportions. For example, oligonucleotide analogs can contain non-naturallyoccurring portions, such as altered sugar moieties or inter-sugarlinkages, such as a phosphorothioate oligodeoxynucleotide. Functionalanalogs of naturally occurring polynucleotides can bind to RNA or DNA,and include peptide nucleic acid (PNA) molecules.

In several examples, oligonucleotides and oligonucleotide analogs caninclude linear sequences up to about 200 nucleotides in length, forexample a sequence (such as DNA or RNA) that is at least 6 bases, forexample at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, or even 200bases long, or from about 6 to about 70 bases, for example about 10-25bases, such as 12, 15, or 20 bases.

Phased: As applied to a sequence of nucleotide pairs for two or morepolymorphic sites in a locus, phased means the combination ofnucleotides present at those polymorphic sites on a copy of the DNA forthe locus.

Polymorphism: A variation in a gene sequence. The polymorphisms can bethose variations (DNA sequence differences) which are generally foundbetween individuals or different ethnic groups and geographic locationswhich, while having a different sequence, produce functionallyequivalent gene products. Typically, the term can also refer to variantsin the sequence which can lead to gene products that are notfunctionally equivalent. Polymorphisms also encompass variations whichcan be classified as alleles and/or mutations which can produce geneproducts which may have an altered function. Polymorphisms alsoencompass variations which can be classified as alleles and/or mutationswhich either produce no gene product or an inactive gene product or anactive gene product produced at an abnormal rate or in an inappropriatetissue or in response to an inappropriate stimulus. Alleles are thealternate forms that occur at the polymorphism.

Polymorphisms can be referred to, for instance, by the nucleotideposition at which the variation exists, by the change in amino acidsequence caused by the nucleotide variation, or by a change in someother characteristic of the nucleic acid molecule or protein that islinked to the variation.

In the instant application “polymorphism” refers a traditionaldefinition, in that the definition “polymorphism” means that the minorallele frequency must be greater than at least 1%.

A “single nucleotide polymorphism (SNP)” is a single base (nucleotide)polymorphism in a DNA sequence among individuals in a population.Typically in the literature, a single nucleotide polymorphism (SNP) mayfall within coding sequences of genes, non-coding regions of genes, orin the intergenic regions between genes. SNPs within a coding sequencewill not necessarily change the amino acid sequence of the protein thatis produced, due to degeneracy of the genetic code. A SNP in which bothforms lead to the same polypeptide sequence is termed “synonymous”(sometimes called a silent mutation). If a different polypeptidesequence is produced they are “nonsynonymous.” A nonsynonymous changemay either be missense or “nonsense,” where a missense change results ina different amino acid, while a nonsense change results in a prematurestop codon.

A tag SNP is a SNP that by itself or in combination with additional tagSNPs indicates the presence of a specific haplotype, or of one member ofa group of haplotypes. The haplotype or haplotypes can indicate agenetic factor is associated with risk for disease, thus a tag SNP orcombination of tag SNPs indicates the presence or absence of riskfactors for disease. A “tag SNP” is a representative single nucleotidepolymorphism (SNP) in a region of the genome with high linkagedisequilibrium (the non-random association of alleles at two or moreloci) that is associated with a disease, such as renal disease, forexample FSGS or ESKD. A tag SNP can be used to identify other SNPs, suchas those with a specified r² value from the tag SNP, which areassociated with a disease, such as FSGS or ESKD. Statistical methods toidentify a tag SNP are known (see Hoperin et al., Bioinformatics 21(suppl): i195-i203, 2005, herein incorporated by reference).

Predictive power: A characteristic for a dichotomous test (one that willreturn either a positive or negative result), indicating increased riskwith a positive result. Predictive power is measured by sensitivity andspecificity. In some examples, the sensitivity of a test is the fractionof people who tested positive for the presence of at least one APOL1risk allele who will develop renal disease, such as FSGS or hypertensiveESKD and the specificity is the fraction of people who tested negativefor the presence of at least one APOL1 risk allele (e.g., absence of atleast one risk allele) who will not develop renal disease. A measure ofthe predictive power of a test is the receiver operator characteristic(ROC) C statistic. The ROC C statistic may be defined as the probability(or fraction of the time) that an individual with the condition has arisk score larger than of an individual without the condition. For atest with no predictive power, the C statistic will be 0.5; for adichotomous test that can invariably correctly identify positives andnegatives (perfect predictive power), the C statistic will be 1.

Probes and primers: Isolated nucleic acids of at least ten nucleotidescapable of hybridizing to a target nucleic acid. A detectable label orreporter molecule can be attached to a probe or primer. Typical labelsinclude radioactive isotopes, enzyme substrates, co-factors, ligands,chemiluminescent or fluorescent agents, haptens, and enzymes. Methodsfor labeling and guidance in the choice of labels appropriate forvarious purposes are discussed, for example in Sambrook et al. (InMolecular Cloning: A Laboratory Manual, CSHL, New York, 1989) andAusubel et al. (In Current Protocols in Molecular Biology, John Wiley &Sons, New York, 1998).

In a particular example, a probe or primer includes at least onefluorophore, such as an acceptor fluorophore or donor fluorophore. Forexample, a fluorophore can be attached at the 5′- or 3′-end of theprobe/primer. In specific examples, the fluorophore is attached to thebase at the 5′-end of the probe/primer, the base at its 3′-end, thephosphate group at its 5′-end or a modified base, such as a T internalto the probe/primer.

Probes are generally at least 15 nucleotides in length, such as at least15, at least 16, at least 17, at least 18, at least 19, least 20, atleast 21, at least 22, at least 23, at least 24, at least 25, at least26, at least 27, at least 28, at least 29, at least 30, at least 31, atleast 32, at least 33, at least 34, at least 35, at least 36, at least37, at least 38, at least 39, at least 40, at least 41, at least 42, atleast 43, at least 44, at least 45, at least 46, at least 47, at least48, at least 49, at least 50 at least 51, at least 52, at least 53, atleast 54, at least 55, at least 56, at least 57, at least 58, at least59, at least 60, at least 61, at least 62, at least 63, at least 64, atleast 65, at least 66, at least 67, at least 68, at least 69, at least70, or more contiguous nucleotides complementary to the target nucleicacid molecule, such as 20-70 nucleotides, 20-60 nucleotides, 20-50nucleotides, 20-40 nucleotides, or 20-30 nucleotides.

Primers are short nucleic acid molecules, for instance DNAoligonucleotides are 10 nucleotides or more in length, which can beannealed to a complementary target nucleic acid molecule by nucleic acidhybridization to form a hybrid between the primer and the target nucleicacid strand. A primer can be extended along the target nucleic acidmolecule by a polymerase enzyme. Therefore, primers can be used toamplify a target nucleic acid molecule.

The specificity of a primer increases with its length. Thus, forexample, a primer that includes 30 consecutive nucleotides will annealto a target sequence with a higher specificity than a correspondingprimer of only 15 nucleotides. Thus, to obtain greater specificity,probes and primers can be selected that include at least 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70 or more consecutive nucleotides. Inparticular examples, a primer is at least 15 nucleotides in length, suchas at least 15 contiguous nucleotides complementary to a target nucleicacid molecule. Particular lengths of primers that can be used topractice the methods of the present disclosure include primers having atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 26, at least 27, at least 28, at least 29, at least 30, at least31, at least 32, at least 33, at least 34, at least 35, at least 36, atleast 37, at least 38, at least 39, at least 40, at least 45, at least50, at least 55, at least 60, at least 65, at least 70, or morecontiguous nucleotides complementary to the target nucleic acid moleculeto be amplified, such as a primer of 15-70 nucleotides, 15-60nucleotides, 15-50 nucleotides, or 15-30 nucleotides.

Primer pairs can be used for amplification of a nucleic acid sequence,for example, by PCR, real-time PCR, or other nucleic-acid amplificationmethods known in the art. An “upstream” or “forward” primer is a primer5′ to a reference point on a nucleic acid sequence. A “downstream” or“reverse” primer is a primer 3′ to a reference point on a nucleic acidsequence. In general, at least one forward and one reverse primer areincluded in an amplification reaction.

Nucleic acid probes and primers can be readily prepared based on thenucleic acid molecules provided herein (such as APOL1). It is alsoappropriate to generate probes and primers based on fragments orportions of these disclosed nucleic acid molecules, for instance regionsthat encompass the identified polymorphisms of interest. PCR primerpairs can be derived from a known sequence by using computer programsintended for that purpose such as Primer (Version 0.5, © 1991, WhiteheadInstitute for Biomedical Research, Cambridge, Mass.) or PRIMER EXPRESS®Software (Applied Biosystems, AB, Foster City, Calif.).

Recessive Model: A genetic based model that tests the association ofhaving two risk alleles (e.g. DD) versus having at least one non-riskallele (e.g., Dd or dd). In some examples, it is a genetic based modelthat tests the association of having two copies of a specified alleleversus having at least one copy of the alternate (reference) allele.

Recombinant: A nucleic acid molecule, protein, cell, or organism thatresults from the recombination of genes (e.g., a sequence that is notnaturally occurring or a sequence that is made by an artificialcombination of two otherwise separated segments of sequence), regardlessof whether naturally or artificially induced. This artificialcombination can be accomplished by chemical synthesis or by theartificial manipulation of isolated segments of nucleic acid molecules,such as by genetic engineering techniques.

Reference Allele: A genotype that predominates in a natural populationof organisms that do not have a disease process, such as renal disease,for example FSGS. In some examples, the reference genotype differs frommutant forms. In other examples, the reference allele is the alternativeallele to a specified allele at a specific locus.

Renal Disease (Nephropathy): A disorder that specifically leads todamage of the kidneys. Renal diseases include but are not limited toFSGS, hypertensive ESKD, nephropathy secondary to systemic lupuserythematosus, diabetic nephropathy, hypertensive nephropathy, IgAnephropathy, nephritis, and xanthine oxidase deficiency.

Renal disease can be chronic or acute. Chronic renal disease, or thetype detected with the assays disclosed herein can progress from stage 1to stage 2, stage 3, stage 4 or stage 5. The stages of chronic renaldisease are:

Stage 1: Slightly diminished kidney function; Kidney damage with normalor increased GFR (>90 mL/min/1.73 m2). Kidney damage is defined aspathologic abnormalities or markers of damage, including abnormalitiesin blood or urine test or imaging studies.

Stage 2: Mild reduction in GFR (60-89 mL/min/1.73 m2) with kidneydamage. Kidney damage is defined as pathologic abnormalities or markersof damage, including abnormalities in blood or urine test or imagingstudies.

Stage 3: Moderate reduction in GFR (30-59 mL/min/1.73 m2)

Stage 4: Severe reduction in GFR (15-29 mL/min/1.73 m2)

Stage 5: Established kidney failure (GFR <15 mL/min/1.73 m2, orpermanent renal replacement therapy (RRT)

The disclosed assays can be used to detect renal disease, such as FSGS,at any of these stages, or prior to stage 1.

Risk Allele: A “risk” allele is an allele associated with a particulartype or form of disease. The risk allele identifies a single nucleotidepolymorphism that can be used to detect or determine the risk for adisease, such as FSGS or hypertensive ESKD.

Sample: A sample, such as a biological sample that includes nucleic acidmolecules, is a sample obtained from a subject. As used herein,biological samples include all clinical samples useful for detection ofrenal disease in subjects, including, but not limited to, cells,tissues, and bodily fluids, such as: blood; derivatives and fractions ofblood, such as serum; extracted galls; biopsied or surgically removedtissue, including tissues that are, for example, unfixed, frozen, fixedin formalin and/or embedded in paraffin; tears; milk; skin scrapes;surface washings; urine; sputum; cerebrospinal fluid; prostate fluid;pus; or bone marrow aspirates. In a particular example, a sampleincludes blood obtained from a human subject, such as whole blood orserum. In another particular example, a sample includes buccal cells,for example collected using a swab or by an oral rinse.

Sequence identity/similarity: The identity/similarity between two ormore nucleic acid sequences, or two or more amino acid sequences, isexpressed in terms of the identity or similarity between the sequences.Sequence identity can be measured in terms of percentage identity; thehigher the percentage, the more identical the sequences are. Sequencesimilarity can be measured in terms of percentage similarity (whichtakes into account conservative amino acid substitutions); the higherthe percentage, the more similar the sequences are. Homologs ororthologs of nucleic acid or amino acid sequences possess a relativelyhigh degree of sequence identity/similarity when aligned using standardmethods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biological Information (NCBI, NationalLibrary of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) andon the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. Additionalinformation can be found at the NCBI web site.

BLASTN is used to compare nucleic acid sequences, while BLASTP is usedto compare amino acid sequences. To compare two nucleic acid sequences,the options can be set as follows: -i is set to a file containing thefirst nucleic acid sequence to be compared (such as C:\seq1.txt); -j isset to a file containing the second nucleic acid sequence to be compared(such as C:\seq2.txt); -p is set to blastn; -o is set to any desiredfile name (such as C:\output.txt); -q is set to -l; -r is set to 2; andall other options are left at their default setting. For example, thefollowing command can be used to generate an output file containing acomparison between two sequences: C:\Bl2seq c:\seq1.txt -j c:\seq2.txt-p blastn -o c:\output.txt -q -l -r 2.

To compare two amino acid sequences, the options of Bl2seq can be set asfollows: -i is set to a file containing the first amino acid sequence tobe compared (such as C:\seq1.txt); -j is set to a file containing thesecond amino acid sequence to be compared (such as C:\seq2.txt); -p isset to blastp; -o is set to any desired file name (such asC:\output.txt); and all other options are left at their default setting.For example, the following command can be used to generate an outputfile containing a comparison between two amino acid sequences: C:\Bl2seqc:\seq1.txt -j c:\seq2.txt -p blastp -o c:\output.txt. If the twocompared sequences share homology, then the designated output file willpresent those regions of homology as aligned sequences. If the twocompared sequences do not share homology, then the designated outputfile will not present aligned sequences.

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresented in both sequences. The percent sequence identity is determinedby dividing the number of matches either by the length of the sequenceset forth in the identified sequence, or by an articulated length (suchas 100 consecutive nucleotides or amino acid residues from a sequenceset forth in an identified sequence), followed by multiplying theresulting value by 100. For example, a nucleic acid sequence that has1166 matches when aligned with a test sequence having 1154 nucleotidesis 75.0 percent identical to the test sequence (i.e.,1166±1554*100=75.0). The percent sequence identity value is rounded tothe nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 arerounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 arerounded up to 75.2. The length value will always be an integer. Inanother example, a target sequence containing a 20-nucleotide regionthat aligns with 20 consecutive nucleotides from an identified sequenceas follows contains a region that shares 75 percent sequence identity tothat identified sequence (that is, 15÷20*100=75).

One indication that two nucleic acid molecules are closely related isthat the two molecules hybridize to each other under stringentconditions, as described above. Nucleic acid sequences that do not showa high degree of identity may nevertheless encode identical or similar(conserved) amino acid sequences, due to the degeneracy of the geneticcode. Changes in a nucleic acid sequence can be made using thisdegeneracy to produce multiple nucleic acid molecules that all encodesubstantially the same protein. Such homologous nucleic acid sequencescan, for example, possess at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or at least 99% sequence identity(for example to a known APOL1 gene sequence) determined by this method.An alternative (and not necessarily cumulative) indication that twonucleic acid sequences are substantially identical is that thepolypeptide which the first nucleic acid encodes is immunologicallycross reactive with the polypeptide encoded by the second nucleic acid.One of skill in the art will appreciate that the particular sequenceidentity ranges are provided for guidance only.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes human and non-human mammals (such as laboratory or veterinarysubjects).

Therapeutically effective amount: An amount of a therapeutic agent thatalone, or together with one or more additional therapeutic agents,induces the desired response. In one example, the desired response isdecreasing the risk of developing FSGS or decreasing the signs andsymptoms of FSGS. In another example, the desired response isameliorating signs or symptoms associated with a Trypanosoma bruceiinfection, such as sleeping sickness. For example, a therapeuticallyeffective amount of a human APOL1 protein comprising a S342Gsubstitution, a I384M substitution and/or a deletion removing aminoacids N388 and Y389, can be used to decrease symptoms associated withsleeping sickness, such as fever, headache, joint pain, lymph nodeswelling, anemia, confusion, reduced coordination, and disruption of thesleep cycle.

Ideally, a therapeutically effective amount provides a therapeuticeffect without causing a substantial cytotoxic effect in the subject.The preparations disclosed herein are administered in therapeuticallyeffective amounts. In general, a therapeutically effective amount of acomposition administered to a human subject will vary depending upon anumber of factors associated with that subject, for example the overallhealth of the subject, the condition to be treated, or the severity ofthe condition. A therapeutically effective amount of a composition canbe determined by varying the dosage of the product and measuring theresulting therapeutic response. The therapeutically effective amount canbe dependent on the source applied, the subject being treated, theseverity and type of the condition being treated, and the manner ofadministration.

In one example, a desired response is to prevent the development ofFSGS. In another example, a desired response is to delay the developmentor progression of FSGS, for example, by at least about three months, atleast about six months, at least about one year, at least about twoyears, at least about five years, or at least about ten years. Inanother example, a desired response is to decrease the signs andsymptoms of FSGS, such as the neurological symptoms in the limbs orassociated with speaking.

Treatment: With respect to disease, either term includes (1) preventingthe disease, e.g., causing the clinical symptoms of the disease not todevelop in an animal that may be exposed to or predisposed to thedisease but does not yet experience or display symptoms of the disease,(2) inhibiting the disease, e.g., arresting the development of thedisease or one or more of its clinical symptoms, or (3) relieving thedisease, e.g., causing regression of the disease or one or more of itsclinical symptoms. For example, treatment can refer to relieving one ormore symptoms associated with Trypanosoma infection, such as sleepingsickness. Treatment of a disease does not require a total absence ofdisease. For example, a decrease of at least 25% or at least 50% of oneor more of the symptoms or undesired consequences of the disease can besufficient.

Trypanosoma brucei: A parasite that causes African trypanosomiasis(sleeping sickness) in humans and nagana in animals in Africa. Theinsect vector for T. brucei is the tsetse fly. There are threesub-species of T. brucei: T b. brucei, T b. rhodesiense and T b.gambiense. A “disease associated with Trypanosoma infection” includesthose diseases that result from infection with T. brucei, such assleeping sickness. For example, infection by T. brucei gambiense causesslow onset chronic trypanosomiasis in humans while infection by T.brucei rhodesiense causes fast onset acute trypanosomiasis in humans. Tbrucei brucei infection causes animal African trypanosomiasis.

II. Methods for Detecting a Genetic Predisposition to and/or anIncreased Risk of Developing Renal Disease

Methods for determining the genetic predisposition to, or an increasedrisk of, development of a renal disease in a subject are providedherein. Disclosed herein are methods for determining the geneticpredisposition to FSGS, as well as methods for determining the geneticpredisposition of a subject to hypertensive end-stage kidney disease(EKSD). However, the methods disclosed herein also can be used to detectany form of renal disease, such as, but not limited to, FSGS or EKSD.The methods also can be used to determine the risk of developing renaldisease. The methods are also useful in genetic confirmations of adiagnosis of renal disease, or to determine a therapeutic regimen for asubject. The methods are useful not only in determining risk, but forgenetic confirmation of suspected chronic renal disease, for example asubject who presents with a reduced glomerular filtration rate (GFR) orother laboratory evidence of renal impairment (such as elevated bloodurea nitrogen (BUN) or abnormal renal histology), or someone withclinical presentation (symptoms) of renal disease, such as fatigue andliquid retention. The methods disclosed herein can be used to determinethe genetic predisposition, detect, or determine the risk of developingnephropathy secondary to systemic lupus erythematosus and othernephropathies. The methods are also useful for determining a therapeuticregimen in treating a subject of interest, or determining if a subjectwill benefit from treatment with a therapeutic regimen of interest. Inpatients having one or more APOL1 gene risk alleles, treatment while thepatient is asymptomatic may be warranted.

In some embodiments, the method includes detecting the presence of agenotype (e.g., the presence of at least one risk allele), such as atleast one single nucleotide polymorphism (SNP) and/or at least oneinversion in a subject (e.g., one or more of the SNPs described herein,such as the G1 and/or G2 risk alleles, or an inversion in the APOL1gene, such as the G3 allele), such that the presence of the at least oneSNP and/or the at least one inversion determines genetic predispositionto (and/or increased risk of developing) renal disease in the subject.In other embodiments, the method includes detecting the presence of agenotype, such that both alleles of the genotype of the subject are riskalleles that indicate a genetic predisposition to renal disease in thesubject. In other embodiments, the method includes detecting thepresence of a tag SNP. In further embodiments, the method includesdetecting the presence of a genotype, such that one of the alleles ofthe genotype is a risk allele of a tag SNP. In still other embodiments,the method includes determining whether the subject is heterozygous foran APOL1 risk allele (e.g., the subject has one risk and one non-riskallele at a given locus) or whether the subject is homozygous for atleast one APOL1 risk allele (e.g., whether both alleles of the subjectat a given locus include an APOL1 risk allele). In an embodiment of themethod, determination that a subject is heterozygous for an APOL1 riskallele at a given locus indicates the subject has an increased risk ofrenal disease relative to a subject that is homozygous at that locus fora non-risk APOL1 allele, and determination that a subject is homozygousfor an APOL1 risk allele at a given locus indicates the subject has asubstantially increased risk of renal disease relative to a subject thatis homozygous at that locus for a non-risk APOL1 allele.

In some embodiments, the method uses two or more SNPs and/or tag SNPs(alone or in combination with one or more inversions) to identify thepresence in the genome of a subject of one or two or more riskhaplotypes. In some embodiments, both of the haplotypes identified ascarried by the subject are copies of a risk haplotype. In otherembodiments, one of the haplotypes is a risk haplotype.

In some embodiments, the method includes detecting the presence of atleast one SNP and at least one inversion in a gene of interest, forexample, APOL1.

In some embodiments, the methods disclosed herein can be used todetermine the genetic predisposition of a human subject to renaldisease, wherein the subject is of African ancestry, such as anAfrican-American subject (a subject who is of African ancestry whoresides in the United States) or an African-European subject (a subjectwho is of African ancestry who resides in Europe) or a subject ofHispanic ancestry. In additional embodiments, the methods disclosedherein can be used to determine the genetic predisposition of a humansubject to renal disease, wherein the subject is of European ancestry.The human subject can self-identify themselves (such as on aquestionnaire) as being of European ancestry, such as identifyingthemselves as Caucasian. There are a number of programs available toconfirm European ancestry, if such confirmation is desired. Theseinclude the program STRUCTURE™ (available on the internet atpritch.bsd.uchicago.edu/structure.html) and the program EURASIANDNA™,version 1.0 and 2.0 (available from DNAPRINT™). In other embodiments,the subject can self-identify themselves (such as on a questionnaire) asbeing of a specific ancestry. However, there are a number of programsavailable to confirm ancestry, if such confirmation is desired. Theseinclude the program STRUCTURE™ (available on the internet atpritch.bsd.uchicago.edu/structure.html). In several examples, thesubject is infected with a human immunodeficiency virus, such as HIV-1or HIV-2.

In some embodiments, the methods include obtaining a sample includingnucleic acids from a human subject of interest, and analyzing the samplefor the presence of at least one SNP and/or at least one inversion, or ahaplotype including at least one tag SNP in these nucleic acids. Inother embodiments, a sample is obtained that contains nucleic acids froma human subject of interest, and the sample is analyzed for the presenceof a haplotype including at least two tag SNPs in a non-coding region ofa gene of interest. The methods can include selecting a subject in needof detecting the presence of the SNP, and obtaining a sample includingnucleic acids from this subject. For example, a subject can be selectedwho is suspected to possess a genetic predisposition to renal disease,such as FSGS or hypertensive ESKD. In another example, a subject can beselected that is of African ancestry and/or is infected with HIV. In afurther example, a subject can be selected who has renal disease, suchas, but not limited to FSGS or hypertensive ESKD. Thus, the subject'srisk for progressing to another stage of renal disease can be detected.The methods disclosed herein can also be used to confirm the presence ofrenal disease in the subject. In yet another example, a subject withrenal disease is selected to determine if a particular therapeuticregimen is appropriate for the subject. A subject of interest (e.g., anasymptomatic subject) can also be selected for preventative orprophylactic treatment based on the presence of at least one riskallele.

Biological samples include all clinical samples useful for detection ofrenal disease in subjects, such as cells, tissues, and bodily fluids,for example blood; derivatives and fractions of blood, such as serum;extracted galls; biopsied or surgically removed tissue, includingtissues that are, for example, unfixed, frozen, fixed in formalin and/orembedded in paraffin; tears; milk; skin scrapes; surface washings;urine; sputum; cerebrospinal fluid; prostate fluid; pus; or bone marrowaspirates. In a particular example, a sample includes blood obtainedfrom a human subject, such as whole blood or serum. In anotherparticular example, a sample includes buccal cells, for examplecollected using a swab or by an oral rinse. In additional embodiments,the method includes analyzing DNA sequence data previously obtained fromthe subject of interest.

APOL1 SNPs and Inversions

In one example, a method for detecting genetic predisposition to, orincreased risk of developing, a renal disease, such as FSGS orhypertensive ESKD, in a human subject is performed by detecting thepresence of at least one SNP and/or at least one inversion in an APOL1gene (e.g., an inversion that replaces all or a portion of the first,second, and/or third exon of the APOL1 gene with another apolipoproteingene, such as the APOL4 gene). In particular examples, specific SNPs ofuse in identifying a genetic predisposition to renal disease (forexample, in a subject of African ancestry, such as an African-Americansubject) include a G at rs73885319, a G at rs60910145, a 6 bp deletion(−/TTATAA; SEQ ID NO:6) at rs71785313, and/or combinations thereof. Insome examples SNP rs73885319 results in a substitution of glycine forserine at amino acid 342 of an APOL1 protein (S342G). In other examples,SNP rs60910145 results in a substitution of methionine for isoleucine atamino acid 384 of an APOL1 protein (I384). In further examples, SNPrs71785313 results in a deletion of amino acids 388 and 389 of an APOL1protein. In other examples, a specific inversion is the G3 inversiondiscussed below).

The method can also include detecting one of more of the APOL1 SNPsand/or inversions disclosed herein. Thus, the method can includedetecting at least one, at least two, or at least three different SNPsand/or at least one, two, or three different inversions. In someembodiments, the SNPs can be in any combination, of at least twodifferent SNPs. Detection of all of the SNPs disclosed herein can alsobe used to detect a genetic predisposition to renal disease, such asFSGS or hypertensive ESKD.

In several embodiments, at least one SNP is detected in a coding regionof an APOL1 gene. Thus, the method can include detecting at least one,at least two, or at least three different SNPs in the coding region ofan APOL1 gene, wherein at least one or more SNPs in the coding region ofthe gene is a G at rs73885319, a G at rs60910145, and/or a 6 bp deletion(−/TTATAA; SEQ ID NO: 6) at rs71785313. In some examples, a G atrs73885319 includes an APOL1 nucleic acid having a G at nucleotide 1024of SEQ ID NO: 4. In some examples, a G at rs60910145 includes an APOL1nucleic acid having a G at nucleotide 1052 of SEQ ID NO: 4. In someexamples, a 6 bp deletion at rs71785313 includes an APOL1 nucleic acidhaving a deletion of nucleotides 1064-1069 of SEQ ID NO: 4.

With regard to the SNPs, the SNPs can be identified by name. The exactsequence of the SNP can be determined from the database of SNPsavailable at the NCBI website (ncbi.nlm.nih.gov/SNP, Apr. 18, 2010). The“position” of the nucleotide of interest is the location in the genomeof the SNP, referring to the nucleotide position from the p-terminus ofthe chromosome in the human genome, see the NCBI SNP website, availableon the internet. Sequence information for each of the APOL1 SNPs listedabove is provided in Table 1.

TABLE 1 APOL1 single nucleotide polymorphisms Risk Reference SNP alleleallele Flanking sequence rs73885319 G A TCAAGCTCACGGATGTGGCCCCTGTA[G/A]GCTTCTTTCTTGTGCTG GATGTAGT (SEQ ID NO: 1) rs60910145 G TCAGGAGCTGGAGGAGAAGCTAAAC AT[G/T]CTCAACAATAATTATAAGATTCTGC (SEQ ID NO: 2) rs71785313 del6 TTATAA GAGAAGCTAAACATTCTCAACAAT(SEQ ID AA[-/TTATAA]GATTCTGCAGGC NO: 6) GGACCAAGAACTG (SEQ ID NO: 3)

In Table 1, the “risk” allele identifies the SNP that can be used todetect or determine the risk for renal disease, such as FSGS orhypertensive ESKD. The “reference” allele is a different allele notassociated with renal disease, and thus is a “protective allele” as thisallele indicates that the subject does not have or is not at risk fordeveloping renal disease, such as FSGS or hypertensive ESKD. In thesequences provided above, the notation “[X/Y]” is used, wherein one of Xor Y is the risk allele and one of X or Y is the reference (protective)allele. For each sequence, the allele associated with renal disease (the“risk” allele) is listed. The allele that is associated with a decreasedrisk (or absence) of renal disease is also listed (the “reference”allele).

Another risk allele that can be used to detect or determine the risk forrenal disease, such as FSGS or hypertensive ESKD, is an APOL1 genechromosomal rearrangement that inverts a segment of DNA including the 5′end of APOL4, all of APOL2, and the 5′ end of APOL1, which produces anAPOL4/APOL1 hybrid gene. The inversion is referred to herein as the “G3”risk allele.

The disclosed methods can include detecting at least one risk allele(e.g., G1, G2, del6, and/or G3) on one or both chromosomes, detectingthe presence of a protective allele on one or both chromosomes, ordetecting the absence of the protective allele on one or bothchromosomes. In some embodiments, detecting the presence of the riskallele indicates that the subject has a genetic predisposition to renaldisease, and detecting the absence of the protective allele indicatesthat the subject has a genetic predisposition to renal disease.Similarly, detecting the absence of the risk allele indicates that thesubject does not have a genetic predisposition to renal disease, anddetecting the presence of the protective allele indicates that thesubject does not have a genetic predisposition to renal disease.

Thus, the disclosed methods can detect a low risk of developing renaldisease, or identify a subject that does not have a geneticpre-disposition to developing renal disease. For example, subjects thathave at least one SNP associated with the reference allele are notgenetically pre-disposed to developing renal disease, such as FSGS orhypertensive ESKD. These subjects do not have renal disease and/or havea low risk for developing renal disease.

In subjects of African ancestry, the methods include detecting thepresence at least one SNP (e.g., G1, G2, and/or del6) in the last (3′)exon of the APOL1 gene or at least one inversion in an APOL1 gene (e.g.,G3). An exemplary nucleic acid sequence for human apolipoprotein L1 is:

(SEQ ID NO: 4) atggagggag ctgctttgct gagagtctct gtcctctgcatctggatgag tgcacttttc cttggtgtgg gagtgagggcagaggaagct ggagcgaggg tgcaacaaaa cgttccaagtgggacagata ctggagatcc tcaaagtaag cccctcggtgactgggctgc tggcaccatg gacccagaga gcagtatctttattgaggat gccattaagt atttcaagga aaaagtgagcacacagaatc tgctactcct gctgactgat aatgaggcctggaacggatt cgtggctgct gctgaactgc ccaggaatgaggcagatgag ctccgtaaag ctctggacaa ccttgcaagacaaatgatca tgaaagacaa aaactggcac gataaaggccagcagtacag aaactggttt ctgaaagagt ttcctcggttgaaaagtgag cttgaggata acataagaag gctccgtgcccttgcagatg gggttcagaa ggtccacaaa ggcaccaccatcgccaatgt ggtgtctggc tctctcagca tttcctctggcatcctgacc ctcgtcggca tgggtctggc acccttcacagagggaggca gccttgtact cttggaacct gggatggagttgggaatcac agccgctttg accgggatta ccagcagtaccatggactac ggaaagaagt ggtggacaca agcccaagcccacgacctgg tcatcaaaag ccttgacaaa ttgaaggaggtgagggagtt tttgggtgag aacatatcca actttctttccttagctggc aatacttacc aactcacacg aggcattgggaaggacatcc gtgccctcag acgagccaga gccaatcttcagtcagtacc gcatgcctca gcctcacgcc cccgggtcactgagccaatc tcagctgaaa gcggtgaaca ggtggagagggttaatgaac ccagcatcct ggaaatgagc agaggagtcaagctcacgga tgtggcccct gtaagcttct ttcttgtgctggatgtagtc tacctcgtgt acgaatcaaa gcacttacatgagggggcaa agtcagagac agctgaggag ctgaagaaggtggctcagga gctggaggag aagctaaaca ttctcaacaataattataag attctgcagg cggaccaaga actgtga

An exemplary amino acid sequence for human apolipoprotein L1 is:

(SEQ ID NO: 5) megaallrvs vlciwmsalf lgvgvraeea garvqqnvpsgtdtgdpqsk plgdwaagtm dpessified aikyfkekvstqnllllltd neawngfvaa aelprneade lrkaldnlarqmimkdknwh dkgqqyrnwf lkefprlkse lednirrlraladgvqkvhk gttianvvsg slsissgilt lvgmglapfteggslvllep gmelgitaal tgitsstmdy gkkwwtqaqahdlviksldk lkevreflge nisnflslag ntyqltrgigkdiralrrar anlqsvphas asrprvtepi saesgeqvervnepsilems rgvkltdvap vsfflvldvv ylvyeskhlhegaksetaee lkkvagelee klnilnnnyk ilqadgel

Methods are also disclosed for detection of a genetic predisposition torenal disease, such as FSGS or hypertensive ESKD, or both in a humansubject of European ancestry. The assay can be used for early diagnosis,for example before the development of renal insufficiency or renalfailure, or for confirming the diagnosis of renal disease. The presenceof at least one SNP or at least one inversion in an APOL1 gene thatencodes apolipoprotein L1 determines the genetic predisposition to FSGSor hypertensive ESKD or both in the human subject of European ancestry.In one embodiment, the method includes detecting at least one of a G atrs73885319, a G at rs60910145, and/or a 6 bp deletion (−/TTATAA; SEQ IDNO: 6) at rs71785313 and/or at least one inversion. In a furtherexample, the method includes detecting the absence of at least one of aG at rs73885319, a G at rs60910145, and/or a 6 bp deletion (−/TTATAA;SEQ ID NO: 6) at rs71785313 and/or at least one inversion. In someexamples, a G at rs73885319 includes an APOL1 nucleic acid having a G atnucleotide 1024 of SEQ ID NO: 4. In some examples, a G at rs60910145includes an APOL1 nucleic acid having a G at nucleotide 1052 of SEQ IDNO: 4. In some examples, a 6 bp deletion at rs71785313 includes an APOL1nucleic acid having a deletion of nucleotides 1064-1069 of SEQ ID NO: 4.

In a further embodiment, the frequency of the risk allele in subjects ofAfrican ancestry is at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40% or at least50%. In several instances, the SNP and/or inversion is in a codingregion of an APOL1 gene. In several embodiments, the SNP used toidentify the frequency of the risk allele in subjects of Africanancestry is set forth in Table 1 and also may include at least oneinversion. In one embodiment, the subject of African ancestry isAfrican-American.

In other embodiments, the risk of renal disease in a subject of Africanor Hispanic ancestry increases if the subject has at least one riskallele (e.g., at least one of a G1, G2, del6, and/or G3 allele).Subjects of African or Hispanic ancestry that have at least two (ormore) of APOL1 gene risk alleles exhibit a significantly increased riskof developing renal disease. The methods of the invention may furtherinclude assaying the subject for the presence of a wild type allele(relative to an APOL1 gene risk allele) as a means for determiningwhether the subject has a moderate or increased risk of renal disease.For example, a subject that is heterozygous at a given locus for one ormore of the APOL1 gene risk alleles may have a greater risk of renaldisease relative to a subject lacking any APOL1 gene risk alleles. Asubject that is homozygous at a given locus for one or more APOL1 generisk alleles may have a risk of renal disease that is greater than thatof a subject that is heterozygous for an APOL1 gene risk allele at thatlocus and a subject that lacks any risk alleles in an APOL1 gene. Thepresence of two or more (e.g., three, four, or more) risk alleles atdifferent loci further increases the likelihood of renal disease in asubject.

In other embodiments, a subject having one or more (e.g., two, three, orfour or more) APOL1 gene risk alleles (e.g., at least one SNP, e.g., G1,G2, and/or del6, and/or at least one inversion, such as the G3, riskallele; the subject may also be heterozygous or homozygous for one ormore of these risk alleles) may be offered a treatment regimen that isdifferent from that of a subject having no or only one APOL1 gene riskalleles. For example, a subject having one or more APOL1 gene riskalleles may be treated with a medication or therapy to reduce or preventrenal disease while the subject is asymptomatic (e.g., the subject maybe subjected to a change in diet, an increase in exercise, a reductionin the intake of NSAIDs, a regimen of blood pressure medication(s) (seelist below) that do not produce a renal toxicity profile, hemodialysis,peritoneal dialysis, or transplantation). The treatment of such apatient may begin at a time point that is earlier than that for asubject having no or only one APOL1 gene risk allele; the amount ofmedication that is prescribed to such a patient may be increased ordecreased in order to avoid further harm to the kidneys; or the type(s)of medication(s) may be adjusted, relative to a subject having no oronly one APOL1 risk allele.

In other embodiments, a subject having one or more APOL1 gene riskalleles may be offered a treatment regimen with respect to bloodpressure medications, steroids, and/or immunosuppressive agents, that isdifferent from a subject lacking any (or only having, e.g., one) APOL1gene risk allele. In particular, subjects having one or more APOL1 generisk alleles are more susceptible to renal damage and/or disease and therisk of kidney damage increases in patients having one or more APOL1gene risk alleles that are treated with blood pressure medications,steroids, and/or immunosuppressive agents that exhibit renal toxic sideeffects. Thus, in patients having one or more APOL1 gene risk alleles,the concentration of a given blood pressure medication, steroid, and/orimmunosuppressive agent and/or the length of treatment may be decreasedrelative to a patient lacking any (or having only one) APOL1 gene riskalleles to avoid damage to the patient's kidneys. The change intherapeutic regimen in patients having one or more APOL1 gene riskalleles may occur while the patients are asymptomatic.

Examples of therapeutics include blood pressure medications (e.g., adiuretic (e.g., chlorthalidone, chlorothiazide, furosemide,hydrochlorothiazide, indapamide, metolazone, amiloride hydrochloride,spironolactone, triamterene, bumetanide, or a combination thereof), analpha adrenergic antagonist (e.g., alfuzosin, doxazosin, prazosin,terazosin, or tamsulosin, or a combination thereof), a centraladrenergic inhibitor (e.g., clonidine, guanfacine, or methyldopa, or acombination thereof), an angiotensin converting enzyme (ACE) inhibitor(e.g., benazepril, captopril, enalapril, fosinopril, lisinopril,moexipril, perindopril, quinapril, ramipril, or trandolapril, orcombinations thereof), an angiotensin II receptor blocker (e.g.,candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan,or valsartan, or combinations thereof), an alpha blocker (e.g.,doxazosin, prazosin, or terazosin, or a combination thereof), a betablocker (e.g., acebutolol, atenolol, betaxolol, bisoprolol, carteolol,metoprolol, nadolol, nebivolol, penbutolol, pindolol, propranolol,solotol, or timolol, or a combination thereof), a calcium channelblocker (e.g., amlodipine, bepridil, diltiazem, felodipine, isradipine,nicardipine, nifedipine, nisoldipine, or verapamil, or combinationthereof), a vasodilator (e.g., hydralazine or minoxidil, or combinationthereof), and a renin inhibitor (e.g., aliskiren), or combinationsthereof), a steroid (e.g., a corticosteroid, such as cortisone,prednisone, methylprednisolone, or prednisolone), or an anabolic steroid(anatrofin, anaxvar, annadrol, bolasterone, decadiabolin, decadurabolin,dehydropiandrosterone (DHEA), delatestryl, dianiabol, dihydrolone,durabolin, dymethazine, enoltestovis, equipose, gamma hydroxybutyrate,maxibolin, methatriol, methyltestosterone, parabolin, primobolin,quinolone, therabolin, trophobolene, and winstrol), or animmunosuppressive agent, such as a glucocorticoid, a cytostatic, anantibody, or an anti-immunophilin and/or mychophenolate mofetil (MMF),FK-506, azathioprine, cyclophosphamide, methotrexate, dactinomycin,antithymocyte globulin (ATGAM), an anti-CD20-antibody, a muromonoab-CD3antibody, basilizimab, daclizumab, cyclosporin, tacrolimus, voclosporin,sirolimus, an interferon, infliximab, etanercept, adalimumab,fingolimod, and/or myriocin).

Subjects having African ancestry (including some subjects of Hispanicancestry) exhibit a 35-45% increased risk of renal disease when thatsubject is determined to have at least one APOL1 gene risk allele (e.g.,G1, G2, del6, and or G3). The risk of FSGS increases by 10-fold in thesesubjects. Surprisingly, the risk of HIV-associated nephropathy increasesby 50-fold in subjects having at least one risk allele. In addition, therisk of ESKD increases by 7-8 fold in subjects having at least one riskallele. These risk factors are not seen in non-African patients lackingone or more of these risk alleles.

In a typical population of subjects of African ancestry, at least 10-15%of the population is at high risk of renal disease due to the presenceof one or more risk alleles. Thirty percent of these subjects are atslight or increased risk, while 55% are at low risk of renal disease.Those subjects having two risk alleles are at the greatest risk of renaldisease. The rate of renal disease in subjects of non-African ancestryis essentially the same for subjects of African ancestry with 0 or, insome instances, 1 risk allele. Thus, the presence of APOL1 risk allelesaccount for most of the large increase in renal disease risk in blackcompared to white individuals.

Kidney Transplantation

A subject in need of kidney transplantation can also be genotyped forthe presence of at least one risk allele in the APOL1 gene disclosedherein. It is known that individuals of African ancestry, includingthose individuals of Hispanic ancestry and, in particular,African-Americans, have an elevated risk for carrying one or two copiesof at least one risk allele the APOL1 gene, which increases their riskof developing idiopathic kidney disease. Thus, in one embodiment, akidney recipient can be genotyped to determine if the recipient carriesone or two copies of at least one of the disclosed risk alleles theAPOL1 gene. Additionally, a kidney selected for transplantation canundergo genotyping prior to surgery to establish the genotype status ofthe organ.

In some embodiments, if the recipient is negative for risk alleles inthe APOL1 gene and the donor kidney is positive for risk alleles in theAPOL1 gene, then the recipient is given pre- and/or post-transplantationtreatment regimens that reduce the risk of the donated kidney undergoingsubsequent kidney failure. Additionally, it may be necessary to treat asubject who is to receive a kidney that is positive for one or more riskalleles in the APOL1 gene differently from a subject who is to receive akidney that does not possess an APOL1 risk allele. Therapeutic treatmentand regimens can therefore be developed after genotyping of a subject oran organ for APOL1 genotype. These treatment regimens may includedecreasing the dosage of, or the length of treatment with, one or moretherapeutics in those individuals having at least one (e.g., two ormore) risk alleles. These therapeutics include blood pressuremedications, steroids, and immunosuppressive agents (see list above).

In other embodiments, the determination that a potential transplantationdonor has one or more risk alleles in the APOL1 gene (e.g., at least onerisk allele at a given locus on one or both chromosomes) indicates thatan organ (e.g., a kidney) of the donor is not suitable or has a lowersuitability for transplant into a recipient relative to a potentialtransplant donor that lacks one or more risk alleles in the APOL1 gene(e.g., at least one risk allele at a given locus on one or bothchromosomes).

III. Methods for Identifying Resistance to Infection by Trypanosoma

APOL1 is a trypanolytic factor of human serum (Vanhamme et al., Nature422:83-87, 2003; Perez-Morga et al., Science 309:469-472, 2005). TheAPOL1 variants disclosed herein exhibit the ability to kill Trypanosomabrucei, the parasite responsible for sleeping sickness disease.Therefore, the disclosed APOL1 variants can be used to detect resistanceof a subject (for example, a mammal, such as a human subject) to adisease associated with Trypanosoma infection.

Trypanosoma brucei is a heterotrophic species from the Trypanosomagenus. It exists in two forms: an insect vector, and once inside thebloodstream, a mammalian host. T. brucei exists as its insect vector inthe tsetse fly, a large, biting fly originating in Africa. Once thetsetse fly bites a mammal, the microbe enters the bloodstream where ittransforms into the mammalian host form, and is then capable of mutatingand invading the central nervous system, (CNS). Once inside the CNS, ithas the ability to inflict African trypanosomiasis, (sleeping sickness).

There are three sub-species of T. brucei: T b. brucei, T b. gambiense,and T b. rhodesiense. T b. gambiense causes slow onset chronictrypanosomiasis in humans. It is most common in central and westernAfrica, where humans are thought to be the primary reservoir. T. bruceirhodesiense causes fast onset acute trypanosomiasis in humans and ismost common in southern and eastern Africa, where game animals andlivestock are thought to be the primary reservoir. T. brucei bruceicauses animal African trypanosomiasis. T b. brucei is generally nothuman infective due to its susceptibility to lysis by humanapolipoprotein L1. T b. gambiense parasites can further be divided intotwo types, type 1, which is homogeneous and clearly distinct from T b.rhodesiense, and type 2, which is heterogeneous and sharescharacteristics with T b. rhodesiense.

In one example, a method for detecting resistance to a diseaseassociated with Trypanosoma (such as sleeping sickness) in a humansubject is performed by detecting the presence of at least one SNP or atleast one inversion in an APOL1 gene (e.g., G1, G2, del6, and/or G3). Inparticular examples, specific SNPs of use in identifying resistance to adisease associated with Trypanosoma (for example, in a subject ofAfrican ancestry) include a G at rs73885319, a G at rs60910145, a 6 bpdeletion (−/TTATAA; SEQ ID NO: 6) at rs71785313, and combinationsthereof. In some examples SNP rs73885319 results in a substitution ofglycine for serine at amino acid 342 of an APOL1 protein (S342G). Inother examples, SNP rs60910145 results in a substitution of methioninefor isoleucine at amino acid 384 of an APOL1 protein (I384). In furtherexamples, SNP rs71785313 results in a deletion of amino acids 388 and389 of an APOL1 protein.

The method can also include detecting one of more of the APOL1 SNPs orinversions disclosed herein. Thus, the method can include detecting atleast one, at least two, or at least three different SNPs (such as 1, 2,or 3 SNPs or inversions). In some embodiments, the SNPs and/or inversioncan be in any combination (e.g., a combination of at least two differentSNPs alone or in combination with an inversion). Detection of one ormore (e. g., all) of the SNPs and/or the inversions disclosed herein canalso be used to detect resistance to a disease associated withTrypanosoma infection (e.g., G1, G2, del6, and/or G3).

In several embodiments, at least one SNP and/or at least one inversionis detected in a coding region of an APOL1 gene. Thus, the method caninclude detecting at least one, at least two, or at least threedifferent SNPs and/or inversions in the coding region of an APOL1 gene,wherein at least one or more SNPs in the coding region of the gene is aG at rs73885319, a G at rs60910145, or a 6 bp deletion (−/TTATAA; SEQ IDNO: 6) at rs71785313, and/or one of the inversions is G3. Sequenceinformation for each of the APOL1 SNPs listed above is provided in Table2.

TABLE 2 APOL1 single nucleotide polymorphisms Resistance Reference   SNPallele allele  Flanking sequence rs73885319 G A TCAAGCTCACGGATGTGG CCCCTGTA[G/A]GCTTC  TTTCTTGTGCTGGATGTA GT (SEQ ID NO: 1) rs60910145 G TCAGGAGCTGGAGGAGAAG  CTAAACAT[G/T]CTCAA  CAATAATTATAAGATTCTGC (SEQ ID NO: 2) rs71785313 del6 TTATAA GAGAAGCTAAACATTCTC (SEQ IDAACAATAA[-/TTATAA]  NO: 6) GATTCTGCAGGCGGACCA  AGAACTG (SEQ ID NO: 3)

In Table 2, the “resistance” allele identifies the SNP that can be usedto detect or determine resistance to a disease associated withTrypanosoma infection, such as sleeping sickness. The “reference” alleleis a different allele not associated with disease resistance. In thesequences provided above, the notation “[X/Y]” is used, wherein one of Xor Y is the resistance allele and one of X or Y is the reference allele.For each sequence, the allele associated with resistance to diseaseassociated with Trypanosoma infection (the “resistance” allele) islisted. The allele that is not associated with a resistance to diseaseis also listed (the “reference” allele).

The disclosed methods can include detecting the resistance allele on oneor both chromosomes, detecting the presence of a reference allele on oneor both chromosomes, or detecting the absence of the resistance alleleon one or both chromosomes. In some embodiments, detecting the presenceof the resistance allele indicates that the subject has a resistance todisease associated with Trypanosoma infection, and detecting the absenceof the reference allele indicates that the subject has a resistance todisease associated with Trypanosoma infection. In particular examples,detecting the presence of the resistance allele indicates that thesubject has a resistance to disease associated with T b. rhodesienseinfection (such as disease associated with infection with type 1 T b.rhodesiense or type 2 T b. rhodesiense). Similarly, detecting theabsence of the resistance allele indicates that the subject does nothave a resistance to disease associated with Trypanosoma infection (suchas disease associated with infection with type 1 T b. rhodesiense ortype 2 T b. rhodesiense), and detecting the presence of the referenceallele indicates that the subject does not have a resistance to diseaseassociated with Trypanosoma infection.

Thus, the disclosed methods can detect resistance to disease associatedwith Trypanosoma infection, such as decreased risk of developingTrypanosoma-associated disease, or identify a subject that does not havea resistance to disease associated with Trypanosoma infection. Forexample, subjects that have at least one APOL1 SNP associated with theresistance allele are genetically pre-disposed to resistance to diseaseassociated with Trypanosoma infection. In particular examples, thesubject is of African or Hispanic ancestry. In further examples, thesubject is African-American.

Methods are also disclosed for detection of a resistance to diseaseassociated with Trypanosoma infection in a human subject of Europeanancestry. The presence of at least one SNP in an APOL1 gene that encodesapolipoprotein L1 determines the genetic predisposition to resistance todisease associated with Trypanosoma infection in the human subject ofEuropean ancestry. In one embodiment, the method includes detecting atleast one of a G at rs73885319, a G at rs60910145, and/or a 6 bpdeletion (−/TTATAA; SEQ ID NO: 6) at rs71785313, and/or at least oneinversion in the APOL1 gene (e.g., G3). In a further example, the methodincludes detecting the absence of at least one of a G at rs73885319, a Gat rs60910145, and/or a 6 bp deletion (−/TTATAA; SEQ ID NO: 6) atrs71785313 and/or at least at least one inversion in the APOL1 gene(e.g., G3).

IV. Methods and Compositions for Treating Disease Associated withTrypanosoma Infection

It has been discovered that human plasma from individuals expressingvariant APOL1 proteins (for example, S342G/I384M and/or del N388/Y389and/or the G3 inversion) lyses Trypanosoma brucei parasites (such asSRA- or SRA+T. brucei) in vitro. Therefore, disclosed herein are methodsfor treating a subject infected with Trypanosoma brucei (such as T b.brucei, T b. rhodesiense, or T b. gambiense) utilizing the variant APOL1proteins described herein. In some embodiments, the method includesadministering to a subject a therapeutically effective amount of avariant APOL1 protein, such as an APOL1 protein with a S342Gsubstitution, an I384M substitution and/or a deletion removing aminoacids N388 and Y389 and/or an APOL1 with a G3 inversion. For example, atherapeutically effective amount of a human APOL1 protein including 1,2, 3 or all 4 of these mutations can be used. For example, atherapeutically effective amount of a human APOL1 protein including aS342G substitution, an I384M substitution and/or a deletion removingamino acids N388 and Y389 and/or an APOL1 with a G3 inversion, can beused to decrease symptoms associated with sleeping sickness, such asfever, headache, joint pain, lymph node swelling, anemia, confusion,reduced coordination, and disruption of the sleep cycle. In particularexamples, the subject is infected with T b. rhodesiense (for example,type 1 T b. rhodesiense or type 2 T b. rhodesiense).

A subject infected with T. brucei is identified by standard diagnosticmethods. In some examples, diagnosis includes demonstrating presence oftrypanosomes in the subject, for example by microscopic examination ofchancre fluid, lymph node aspirates, blood, bone marrow, or, in the latestages of infection, cerebrospinal fluid. In some examples, a wetpreparation is examined for motile trypanosomes and a smear is fixed,stained with Giemsa (or Field), and examined. In other examples, aserological test is used to detect presence of anti-trypanosomeantibodies. Particular serological tests include agglutination tests,such as micro-CATT, wb-CATT, and wb-LATEX (e.g., Truc et al., Bull.World Health Org. 80:882-886, 2002). In further examples, a diagnosis isbased on clinical symptoms, including non-specific symptoms (such asfever, fatigue, headache, arthralgia, and pruritus), enlarged cervicallymph nodes in the posterior cervical triangle (Winterbottom's sign),and neuropsychiatric symptoms and signs (such as mental disturbance,disturbance of the sleep-wake cycle, rigidity and tremor, dysarthria,and ataxia).

Disclosed herein are methods of treating a subject infected with T.brucei which include administering to the subject a therapeuticallyeffective amount of a variant APOL1 protein, such as an APOL1 proteinincluding a S342G substitution, an I384M substitution and/or a deletionremoving amino acids N388 and Y389, and/or an APOL1 with a G3 inversion.In some embodiments, the method includes administering a therapeuticallyeffective amount of human serum or HDL particles including at least oneAPOL1 variant protein (such as an APOL1 protein including a S342Gsubstitution, an I384M substitution and/or a deletion removing aminoacids N388 and Y389, and/or an APOL1 protein with a G3 inversion).

Appropriate human donors for obtaining human serum or HDL particlescontaining APOL1 variant protein can be identified utilizing thegenotyping methods described herein. In some examples, a donor is anindividual with an APOL1 gene having at least one of a G at rs73885319,a G at rs60910145, and/or a 6 base pair deletion at rs71785313 and/or anAPOL1 gene with a G3 inversion.

In some examples, a therapeutically effective amount of human serumincludes at least a 10-fold dilution of serum from a donor with an APOL1protein including a S342G substitution, an I384M substitution and/or adeletion removing amino acids N388 and Y389 and/or an APOL1 protein witha G3 inversion (such as at least a 100-fold, 1000-fold, 10,000-fold,100,000-fold or more dilution). It will be appreciated that thesedosages are examples only, and an appropriate dose can be determined byone of ordinary skill in the art using only routine experimentation.

In other embodiments, the method includes administering atherapeutically effective amount of a recombinant APOL1 protein,including at least one APOL1 variant (such as an APOL1 protein includinga S342G substitution, an I384M substitution and/or a deletion removingamino acids N388 and Y389 and/or an APOL1 protein with a G3 inversion.).In some examples, a therapeutically effective amount of recombinantAPOL1 variant protein includes about 0.1 mg/kg to about 1000 mg/kg (suchas about 1 mg/kg to 1000 mg/kg, about 10 mg/kg to 500 mg/kg, about 10mg/kg to 100 mg/kg, about 50 mg/kg to 500 mg/kg, or about 100 mg/kg to1000 mg/kg). Administration can be accomplished by single or multipledoses. The dose required will vary from subject to subject depending onthe species, age, weight and general condition of the subject, theparticular therapeutic agent being used and its mode of administration.It will be appreciated that these dosages are examples only, and anappropriate dose can be determined by one of ordinary skill in the artusing only routine experimentation.

The preparation of recombinant proteins is well known to those skilledin the art. See, e.g., Sambrook et al. (In Molecular Cloning: ALaboratory Manual, CSHL, New York, 1989); Ausubel et al. (In CurrentProtocols in Molecular Biology, John Wiley & Sons, New York, 1998); andThe Recombinant Protein Handbook, GE Lifesciences, Code 18-1142-75.

Also disclosed herein are pharmaceutical compositions that include avariant APOL1 protein (such as APOL1 protein including a S342G variant,an I384M variant, and/or a del N388/Y389 variant, and/or an APOL1protein with a G3 inversion, or a combination thereof), such as arecombinant APOL1 protein. In some embodiments, the composition includesa pharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are determined in part by the particular composition beingadministered, as well as by the particular method used to administer thecomposition. Accordingly, there is a wide variety of suitableformulations of pharmaceutical compositions of the present disclosure.See, e.g., Remington: The Science and Practice of Pharmacy, TheUniversity of the Sciences in Philadelphia, Editor, Lippincott,Williams, & Wilkins, Philadelphia, Pa., 21^(st) Edition (2005).

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

V. Molecular Methods

Generally, the methods disclosed herein involve an assessment of nucleicacid sequence. Molecular techniques of use in all of these methods aredisclosed below.

Preparation of Nucleic Acids for Analysis: Nucleic acid molecules can beprepared for analysis using any technique known to those skilled in theart. Generally, such techniques result in the production of a nucleicacid molecule sufficiently pure to determine the presence or absence ofone or more variations at one or more locations in the nucleic acidmolecule. Such techniques are described for example, in Sambrook, etal., Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory, New York) (1989), and Ausubel, et al., Current Protocols inMolecular Biology (John Wiley and Sons, New York) (1997), incorporatedherein by reference.

When the nucleic acid of interest is present in a cell, it can benecessary to first prepare an extract of the cell and then performfurther steps, such as differential precipitation, columnchromatography, extraction with organic solvents and the like, in orderto obtain a sufficiently pure preparation of nucleic acid. Extracts canbe prepared using standard techniques in the art, for example, bychemical or mechanical lysis of the cell. Extracts then can be furthertreated, for example, by filtration and/or centrifugation and/or withchaotropic salts such as guanidinium isothiocyanate or urea or withorganic solvents such as phenol and/or chloroform to denature anycontaminating and potentially interfering proteins. When chaotropicsalts are used, it can be desirable to remove the salts from the nucleicacid-containing sample. This can be accomplished using standardtechniques in the art such as precipitation, filtration, size exclusionchromatography and the like. In some examples, nucleic acids can beisolated using commercially available kits (e.g., Qiagen, Valencia,Calif.; Life Technologies/Invitrogen, Carlsbad, Calif.; Epicentre,Madison, Wis.).

In some instances, messenger RNA can be extracted from cells. Techniquesand material for this purpose are known to those skilled in the art andcan involve the use of oligo dT attached to a solid support such as abead or plastic surface. In some embodiments, the mRNA can be reversetranscribed into cDNA using, for example, a reverse transcriptaseenzyme. Suitable enzymes are commercially available from, for example,Life Technologies/Invitrogen (Carlsbad, Calif.). Optionally, cDNAprepared from mRNA can also be amplified.

Amplification of nucleic acid molecules: Optionally, the nucleic acidsamples obtained from the subject are amplified prior to detection.Target nucleic acids are amplified to obtain amplification products,including a SNP or sequences from a haplotype block including a tag SNP,can be amplified from the sample prior to detection. Typically, DNAsequences are amplified, although in some instances RNA sequences can beamplified or converted into cDNA, such as by using RT PCR.

Any nucleic acid amplification method can be used. An example of invitro amplification is the polymerase chain reaction (PCR), in which abiological sample obtained from a subject is contacted with a pair ofoligonucleotide primers, under conditions that allow for hybridizationof the primers to a nucleic acid molecule in the sample. The primers areextended under suitable conditions, dissociated from the template, andthen re-annealed, extended, and dissociated to amplify the number ofcopies of the nucleic acid molecule. Other examples of in vitroamplification techniques include quantitative real-time PCR, stranddisplacement amplification (see U.S. Pat. No. 5,744,311);transcription-free isothermal amplification (see U.S. Pat. No.6,033,881); repair chain reaction amplification (see PCT Publication No.WO 90/01069); ligase chain reaction amplification (see EP-A-320 308);gap filling ligase chain reaction amplification (see U.S. Pat. No.5,427,930); coupled ligase detection and PCR (see U.S. Pat. No.6,027,889); and NASBA™ RNA transcription-free amplification (see U.S.Pat. No. 6,025,134).

In specific examples, the target sequences to be amplified from thesubject include at least one APOL1 SNP, one or more different haplotypeblocks including a tag SNP, or a nucleotide sequence of interestincluding the tag SNP. In certain embodiments, target sequencescontaining one or more of SEQ ID NOs: 1-3, or a subset thereof, areamplified. In an embodiment, a single SNP with exceptionally highpredictive value is amplified, or a nucleic acid encoding the SNP isamplified.

A pair of primers can be utilized in the amplification reaction. One orboth of the primers can be labeled, for example with a detectableradiolabel, fluorophore, or biotin molecule. The pair of primersincludes an upstream primer (which binds 5′ to the downstream primer)and a downstream primer (which binds 3′ to the upstream primer). Thepair of primers used in the amplification reactions are selectiveprimers which permit amplification of a size related marker locus.Primers can be selected to amplify a nucleic acid including a SNP, ahaplotype block including a tag SNP, or a nucleic acid including a tagSNP. Numerous primers can be designed by those of skill in the artsimply by determining the sequence of the desired target region ofAPOL1, for example, using well known computer assisted algorithms thatselect primers within desired parameters suitable for annealing andamplification.

If desired, an additional pair of primers can be included in theamplification reaction as an internal control. For example, theseprimers can be used to amplify a “housekeeping” nucleic acid molecule,and serve to provide confirmation of appropriate amplification. Inanother example, a target nucleic acid molecule including primerhybridization sites can be constructed and included in the amplificationreactor. One of skill in the art will readily be able to identify primerpairs to serve as internal control primers.

Primer Design Strategy: Increased use of polymerase chain reaction (PCR)methods has stimulated the development of many programs to aid in thedesign or selection of oligonucleotides used as primers for PCR. Fourexamples of such programs that are freely available via the Internetare: PRIMER™ by Mark Daly and Steve Lincoln of the Whitehead Institute(UNIX, VMS, DOS, and Macintosh), Oligonucleotide Selection Program byPhil Green and LaDeana Hiller of Washington University in St. Louis(UNIX, VMS, DOS, and Macintosh), PGEN™ by Yoshi (DOS only), and Amplifyby Bill Engels of the University of Wisconsin (Macintosh only).Generally these programs help in the design of PCR primers by searchingfor bits of known repeated-sequence elements and then optimizing theT_(m) by analyzing the length and GC content of a putative primer.Commercial software is also available and primer selection proceduresare rapidly being included in most general sequence analysis packages.

Designing oligonucleotides for use as either sequencing or PCR primersrequires selection of an appropriate sequence that specificallyrecognizes the target APOL1, and then testing the sequence to eliminatethe possibility that the oligonucleotide will have a stable secondarystructure. Inverted repeats in the sequence can be identified using arepeat-identification or RNA-folding programs. If a possible stemstructure is observed, the sequence of the primer can be shifted a fewnucleotides in either direction to minimize the predicted secondarystructure. When the amplified sequence is intended for subsequencecloning, the sequence of the oligonucleotide can also be compared withthe sequences of both strands of the appropriate vector and insert DNA.A sequencing primer only has a single match to the target DNA. It isalso advisable to exclude primers that have only a single mismatch withan undesired target DNA sequence. For PCR primers used to amplifygenomic DNA, the primer sequence can be compared to the sequences in theGENBANK™ database to determine if any significant matches occur. If theoligonucleotide sequence is present in any known DNA sequence or, moreimportantly, in any known repetitive elements, the primer sequenceshould be changed.

Detection of alleles: The nucleic acids obtained from the sample can begenotyped to identify the particular allele present for a marker locus.A sample of sufficient quantity to permit direct detection of markeralleles from the sample can be obtained from the subject. Alternatively,a smaller sample is obtained from the subject and the nucleic acids areamplified prior to detection. Any APOL1 nucleic acid that is informativefor a SNP or inversion or chromosome haplotype can be detected.Generally, the target nucleic acid corresponds to a tag SNP describedabove (SEQ ID NOs: 1-3). Any method of detecting a nucleic acid moleculecan be used, such as hybridization and/or sequencing assays.

Hybridization is the binding of complementary strands of DNA, DNA/RNA,or RNA. Hybridization can occur when primers or probes bind to targetsequences such as target sequences within genomic DNA. Probes andprimers that are useful generally include nucleic acid sequences thathybridize (for example under high stringency conditions) with a nucleicacid sequence including a SNP or inversion of interest, but do nothybridize to a reference allele, or that hybridize to the referenceallele, but do not hybridize to the SNP or inversion. Physical methodsof detecting hybridization or binding of complementary strands ofnucleic acid molecules, include but are not limited to, such methods asDNase I or chemical footprinting, gel shift and affinity cleavageassays, Southern and Northern blotting, dot blotting and lightabsorption detection procedures. The binding between a nucleic acidprimer or probe and its target nucleic acid is frequently characterizedby the temperature (T_(m)) at which 50% of the nucleic acid probe ismelted from its target. A higher (T_(m)) means a stronger or more stablecomplex relative to a complex with a lower (T_(m)).

Generally, complementary nucleic acids form a stable duplex or triplexwhen the strands bind, (hybridize), to each other by formingWatson-Crick, Hoogsteen or reverse Hoogsteen base pairs. Stable bindingoccurs when an oligonucleotide molecule remains detectably bound to atarget nucleic acid sequence under the required conditions.

Complementarity is the degree to which bases in one nucleic acid strandbase pair with the bases in a second nucleic acid strand.Complementarity is conveniently described by percentage, that is, theproportion of nucleotides that form base pairs between two strands orwithin a specific region or domain of two strands. For example, if 10nucleotides of a 15-nucleotide oligonucleotide form base pairs with atargeted region of a DNA molecule, that oligonucleotide is said to have66.67% complementarity to the region of DNA targeted.

In the present disclosure, “sufficient complementarity” means that asufficient number of base pairs exist between an oligonucleotidemolecule and a target nucleic acid sequence (such as a tag SNP) toachieve detectable and specific binding. When expressed or measured bypercentage of base pairs formed, the percentage complementarity thatfulfills this goal can range from as little as about 50% complementarityto full (100%) complementary. In general, sufficient complementarity isat least about 50%, for example at least about 75% complementarity, atleast about 90% complementarity, at least about 95% complementarity, atleast about 98% complementarity, or even at least about 100%complementarity. The qualitative and quantitative considerationsinvolved in establishing binding conditions that allow one skilled inthe art to design appropriate oligonucleotides for use under the desiredconditions is provided by Beltz et al. Methods Enzymol 100:266-285,1983, and by Sambrook et al. (ed.), Molecular Cloning: A LaboratoryManual, 2nd ed., vol. 1-3, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the hybridization method and thecomposition and length of the hybridizing nucleic acid sequences.Generally, the temperature of hybridization and the ionic strength (suchas the Na⁺ concentration) of the hybridization buffer will determine thestringency of hybridization. Calculations regarding hybridizationconditions for attaining particular degrees of stringency are discussedin Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual,second edition, Cold Spring Harbor Laboratory, Plainview, N.Y. (Chapters9 and 11). Exemplary hybridization conditions are provided above.

Methods for labeling nucleic acid molecules so they can be detected arewell known. Examples of such labels include non-radiolabels andradiolabels. Non-radiolabels include, but are not limited to an enzyme,chemiluminescent compound, fluorescent compound (such as FITC, Cy3, andCy5), metal complex, hapten, enzyme, colorimetric agent, a dye, orcombinations thereof. Radiolabels include, but are not limited to, ¹²⁵I,³²P and ³⁵S. For example, radioactive and fluorescent labeling methods,as well as other methods known in the art, are suitable for use with thepresent disclosure. In one example, primers used to amplify thesubject's nucleic acids are labeled (such as with biotin, a radiolabel,or a fluorophore). In another example, amplified target nucleic acidsamples are end-labeled to form labeled amplified material. For example,amplified nucleic acid molecules can be labeled by including labelednucleotides in the amplification reactions.

Nucleic acid molecules corresponding to one or more APOL1 SNPs and/orinversions, or haplotypes blocks, including a tag SNP, can also bedetected by hybridization procedures using a labeled nucleic acid probe,such as a probe that detects only one alternative allele at a markerlocus. Most commonly, the target nucleic acid (or amplified targetnucleic acid) is separated based on size or charge and transferred to asolid support. The solid support (such as membrane made of nylon ornitrocellulose) is contacted with a labeled nucleic acid probe, whichhybridizes to it complementary target under suitable hybridizationconditions to form a hybridization complex.

Hybridization conditions for a given combination of array and targetmaterial can be optimized routinely in an empirical manner close to theT_(m) of the expected duplexes, thereby maximizing the discriminatingpower of the method. For example, the hybridization conditions can beselected to permit discrimination between matched and mismatchedoligonucleotides. Hybridization conditions can be chosen to correspondto those known to be suitable in standard procedures for hybridizationto filters (and optionally for hybridization to arrays). In particular,temperature is controlled to substantially eliminate formation ofduplexes between sequences other than an exactly complementary allele ofthe selected marker. A variety of known hybridization solvents can beemployed, the choice being dependent on considerations known to one ofskill in the art (see U.S. Pat. No. 5,981,185).

Once the target nucleic acid molecules have been hybridized with thelabeled probes, the presence of the hybridization complex can beanalyzed, for example by detecting the complexes.

Methods for detecting hybridized nucleic acid complexes are well knownin the art. In one example, detection includes detecting one or morelabels present on the oligonucleotides, the target (e.g., amplified)sequences, or both. Detection can include treating the hybridizedcomplex with a buffer and/or a conjugating solution to effectconjugation or coupling of the hybridized complex with the detectionlabel, and treating the conjugated, hybridized complex with a detectionreagent. In one example, the conjugating solution includes streptavidinalkaline phosphatase, avidin alkaline phosphatase, or horseradishperoxidase. Specific, non-limiting examples of conjugating solutionsinclude streptavidin alkaline phosphatase, avidin alkaline phosphatase,or horseradish peroxidase. The conjugated, hybridized complex can betreated with a detection reagent. In one example, the detection reagentincludes enzyme-labeled fluorescence reagents or calorimetric reagents.In one specific non-limiting example, the detection reagent isenzyme-labeled fluorescence reagent (ELF) from Molecular Probes, Inc.(Eugene, Oreg.). The hybridized complex can then be placed on adetection device, such as an ultraviolet (UV) transilluminator(manufactured by UVP, Inc. of Upland, Calif.). The signal is developedand the increased signal intensity can be recorded with a recordingdevice, such as a charge coupled device (CCD) camera. In particularexamples, these steps are not performed when radiolabels are used. Inparticular examples, the method further includes quantification, forinstance by determining the amount of hybridization.

Allele Specific PCR: Allele-specific PCR differentiates between targetregions differing in the presence of absence of a variation orpolymorphism. PCR amplification primers are chosen based upon theircomplementarity an APOL1 sequence, such as nucleic acid sequence in aSNP or inversion, haplotype block including a tag SNP, a specifiedregion of an allele including a tag SNP, or to the tag SNP itself. Theprimers bind only to certain alleles of the target sequence. This methodis described by Gibbs, Nucleic Acid Res. 17:12427 2448, 1989, hereinincorporated by reference.

Allele Specific Oligonucleotide Screening Methods: Further screeningmethods employ the allele-specific oligonucleotide (ASO) screeningmethods (e.g. see Saiki et al., Nature 324:163-166, 1986).Oligonucleotides with one or more base pair mismatches are generated forany particular allele or haplotype block. ASO screening methods detectmismatches between one allele (or haplotype block) in the target genomicor PCR amplified DNA and the other allele (or haplotype block), showingdecreased binding of the oligonucleotide relative to the second allele(e.g., the other allele) oligonucleotide. Oligonucleotide probes can bedesigned that under low stringency will bind to both polymorphic formsof the allele, but which at high stringency, only bind to the allele towhich they correspond. Alternatively, stringency conditions can bedevised in which an essentially binary response is obtained. Forexample, an ASO corresponding to a variant form of the target gene willhybridize to that allele (haplotype block), and not to the referenceallele (haplotype block).

Ligase Mediated Allele Detection Method: Ligase can also be used todetect point mutations, such as the SNPs disclosed herein, in a ligationamplification reaction (e.g. as described in Wu et al., Genomics4:560-569, 1989). The ligation amplification reaction (LAR) utilizesamplification of specific DNA sequence using sequential rounds oftemplate dependent ligation (e.g., as described in Wu, supra, andBarany, Proc. Nat. Acad. Sci. 88:189-193, 1990).

Denaturing Gradient Gel Electrophoresis: Amplification productsgenerated using the polymerase chain reaction can be analyzed by the useof denaturing gradient gel electrophoresis. Different SNPs or alleles(haplotype blocks) can be identified based on the differentsequence-dependent melting properties and electrophoretic migration ofDNA in solution. DNA molecules melt in segments, termed melting domains,under conditions of increased temperature or denaturation. Each meltingdomain melts cooperatively at a distinct, base-specific meltingtemperature (T_(m)). Melting domains are at least 20 base pairs inlength, and can be up to several hundred base pairs in length.

Differentiation between SNPs or alleles (haplotype blocks) based onsequence specific melting domain differences can be assessed usingpolyacrylamide gel electrophoresis, as described in Chapter 7 of Erlich,ed., PCR Technology, Principles and Applications for DNA Amplification,W. H. Freeman and Co., New York (1992).

Generally, a target region to be analyzed by denaturing gradient gelelectrophoresis is amplified using PCR primers flanking the targetregion. The amplified PCR product is applied to a polyacrylamide gelwith a linear denaturing gradient as described in Myers et al., Meth.Enzymol. 155:501-527, 1986, and Myers et al., in Genomic Analysis, APractical Approach, K. Davies Ed. IRL Press Limited, Oxford, pp. 95 139,1988. The electrophoresis system is maintained at a temperature slightlybelow the T_(m) of the melting domains of the target sequences.

In an alternative method of denaturing gradient gel electrophoresis, thetarget sequences can be initially attached to a stretch of GCnucleotides, termed a GC clamp, as described in Chapter 7 of Erlich,supra. In one example, at least 80% of the nucleotides in the GC clampare either guanine or cytosine. In another example, the GC clamp is atleast 30 bases long. This method is particularly suited to targetsequences with a high T_(m).

Generally, the target region is amplified by polymerase chain reaction.One of the oligonucleotide PCR primers carries at its 5′ end, the GCclamp region, at least 30 bases of the GC rich sequence, which isincorporated into the 5′ end of the target region during amplification.The resulting amplified target region is run on an electrophoresis gelunder denaturing gradient conditions. DNA fragments differing by asingle base change will migrate through the gel to different positions,which can be visualized by ethidium bromide staining.

Temperature Gradient Gel Electrophoresis: Temperature gradient gelelectrophoresis (TGGE) is based on the same underlying principles asdenaturing gradient gel electrophoresis, except the denaturing gradientis produced by differences in temperature instead of differences in theconcentration of a chemical denaturant. Standard TGGE utilizes anelectrophoresis apparatus with a temperature gradient running along theelectrophoresis path. As samples migrate through a gel with a uniformconcentration of a chemical denaturant, they encounter increasingtemperatures. An alternative method of TGGE, temporal temperaturegradient gel electrophoresis (TTGE or tTGGE) uses a steadily increasingtemperature of the entire electrophoresis gel to achieve the sameresult. As the samples migrate through the gel the temperature of theentire gel increases, leading the samples to encounter increasingtemperature as they migrate through the gel. Preparation of samples,including PCR amplification with incorporation of a GC clamp, andvisualization of products are the same as for denaturing gradient gelelectrophoresis.

Single-Strand Conformation Polymorphism Analysis: Target sequences, suchas alleles or haplotype blocks can be differentiated using single-strandconformation polymorphism analysis, which identifies base differences byalteration in electrophoretic migration of single stranded PCR products,for example as described in Orita et al., Proc. Nat. Acad. Sci.85:2766-2770, 1989. Amplified PCR products can be generated as describedabove, and heated or otherwise denatured, to form single strandedamplification products. Single-stranded nucleic acids can refold or formsecondary structures which are partially dependent on the base sequence.Thus, electrophoretic mobility of single-stranded amplification productscan detect base-sequence difference between alleles or haplotype blocks.

Chemical or Enzymatic Cleavage of Mismatches: Differences between targetsequences, such as alleles or haplotype blocks, can also be detected bydifferential chemical cleavage of mismatched base pairs, for example asdescribed in Grompe et al., Am. J. Hum. Genet. 48:212-222, 1991. Inanother method, differences between target sequences, such as alleles orhaplotype blocks, can be detected by enzymatic cleavage of mismatchedbase pairs, as described in Nelson et al., Nature Genetics 4:11-18,1993. Briefly, genetic material from an animal and an affected familymember can be used to generate mismatch free heterohybrid DNA duplexes.As used herein, “heterohybrid” means a DNA duplex strand comprising onestrand of DNA from one animal, and a second DNA strand from anotheranimal, usually an animal differing in the phenotype for the trait ofinterest. Positive selection for heterohybrids free of mismatches allowsdetermination of small insertions, deletions or other polymorphisms.

Non-gel Systems: Other possible techniques include non-gel systems suchas TaqMan™ (Perkin Elmer). In this system oligonucleotide PCR primersare designed that flank the mutation in question and allow PCRamplification of the region. A third oligonucleotide probe is thendesigned to hybridize to the region containing the base subject tochange between different alleles of the gene. This probe is labeled withfluorescent dyes at both the 5′ and 3′ ends. These dyes are chosen suchthat while in this proximity to each other the fluorescence of one ofthem is quenched by the other and cannot be detected. Extension by TaqDNA polymerase from the PCR primer positioned 5′ on the templaterelative to the probe leads to the cleavage of the dye attached to the5′ end of the annealed probe through the 5′ nuclease activity of the TaqDNA polymerase. This removes the quenching effect allowing detection ofthe fluorescence from the dye at the 3′ end of the probe. Thediscrimination between different DNA sequences arises through the factthat if the hybridization of the probe to the template molecule is notcomplete (there is a mismatch of some form) the cleavage of the dye doesnot take place. Thus only if the nucleotide sequence of theoligonucleotide probe is completely complimentary to the templatemolecule to which it is bound will quenching be removed. A reaction mixcan contain two different probe sequences each designed againstdifferent alleles that might be present thus allowing the detection ofboth alleles in one reaction.

Non-PCR Based Allele detection: The identification of a DNA sequence canbe made without an amplification step, based on polymorphisms includingrestriction fragment length polymorphisms in a subject and a control,such as a family member. Hybridization probes are generallyoligonucleotides which bind through complementary base pairing to all orpart of a target nucleic acid. Probes typically bind target sequenceslacking complete complementarity with the probe sequence depending onthe stringency of the hybridization conditions. The probes can belabeled directly or indirectly, such that by assaying for the presenceor absence of the probe, one can detect the presence or absence of thetarget sequence. Direct labeling methods include radioisotope labeling,such as with ³²P or ³⁵S. Indirect labeling methods include fluorescenttags, biotin complexes which can be bound to avidin or streptavidin, orpeptide or protein tags. Visual detection methods includephotoluminescents, Texas red, rhodamine and its derivatives, red leucodye and 3,3′,5,5′-tetramethylbenzidine (TMB), fluorescein, and itsderivatives, dansyl, umbelliferone and the like or with horse radishperoxidase, alkaline phosphatase and the like.

Hybridization probes include any nucleotide sequence capable ofhybridizing to a nucleic acid sequence wherein a polymorphism is presentthat is associated with FSGS or hypertensive ESKD, such as an APOL1 SNPand/or inversion, or a tag SNP, and thus defining a genetic marker,including a restriction fragment length polymorphism, a hypervariableregion, repetitive element, or a variable number tandem repeat.Hybridization probes can be any gene or a suitable analog. Furthersuitable hybridization probes include exon fragments or portions ofcDNAs or genes known to map to the relevant region of the chromosome.

Exemplary tandem repeat hybridization probes for use in the methodsdisclosed are those that recognize a small number of fragments at aspecific locus at high stringency hybridization conditions, or thatrecognize a larger number of fragments at that locus when the stringencyconditions are lowered.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES Example 1 APOL1 Variants Associated with Focal SegmentalGlomerulosclerosis Methods

FSGS genotype experiment: Variants for the initial FSGS genotype studywere selected by accessing 1000 Genomes Project (1000genomes.org/) datausing the Integrative Genomics Viewer (broadinstitute.org/igv). Allvariants in the region 34,930 kb-35,060 kb (NCBI 36) with estimatedminor allele frequency greater than 15% in Yoruba and minor allelefrequency less than 10% in Europeans were selected, together with someadditional ones with biological relevance, and sent for genotyping usingSequenom technology (sequenom.com). A small amount of SNPs were droppeddue to Multiple eXTEND hits for scanned primer triplets and some failedgenotyping (<20%). Overall three plexes were used for the FSGS analysis.Association of genotype data and association controlling for allelles G1and G2 were performed with plink (pngu.mgh.harvard.edu/˜purcell/plink;Purcell et al., Am. J. Hum. Genet. 81:559-575, 2007) using Fisher'sexact test and logistic regression.

Bounds on causal variant(s): Due to the high frequency differentiationbetween frequency of alleles G1 and G2 in cases and controls, someformal arguments can be made to discard other variants as causal. Definewith A₁ the combined allele G1 and G2 and with A₂ the wild type allele.Define with B₁ the risk version of the combined causal alleles and withB₂ the non-risk version. Assume that in controls the frequency ofhaplotype A₁B₁ is x₁₁, A₁B₂ is x₁₂, A₂B₁ is x₂₁, and A₂B₂ is x₂₂. Definethe frequency in controls of allele A₁ as p₁=x₁₁+x₁₂, and B₁ asq₁=x₁₁+x₂₁. Say p′₁ and q′₁ for the frequencies of A₁ and B₁ in caseswith p′₁>p₁ and q′₁>q₁. Think of x₁₁/q₁ as the fraction of haplotypescontaining B₁ which also contain A₁ and x₁₂/(1−q₁) as the fraction ofhaplotypes containing B₂ which also contain A₁. We can then write:

p ₁=(x ₁₁ /q ₁)q ₁ +x ₁₂/(1−q ₁)(1−q ₁),

p′ ₁=(x ₁₁ /q ₁)q′ ₁ +x ₁₂/(1−q ₁)(1−q′ ₁).

By subtracting one equation from the other:

p′ ₁ −p ₁ =x ₁₁ /q ₁(q′ ₁ −q ₁)+x ₁₂/(1−q ₁)(1−q′ ₁−1+q ₁),

p′ ₁ −p ₁=(x ₁₁ /q ₁ −x ₁₂/(1−q ₁))(q′ ₁ −q ₁).

From this equation, since q′₁<1 and x₁₁<p₁, we get the inequality:

p′ ₁ −p ₁ <p ₁ /q ₁(1−q ₁),

q ₁ <p ₁ /p′ ₁.

In the NIH FSGS cohort, p₁=33% and p′₁=72%, from which we get the boundq₁<46%. The rationale behind this argument is that if the frequency ofthe causal allele is too high in controls, then even if it was 100% incases, this difference would not be able to explain the disparityobserved for alleles in APOL1.Continuing from the previous equation:

p′ ₁ −p ₁=(x ₁₁ −x ₁₁ q ₁ −x ₁₂ q ₁)/(q ₁(1−q ₁))(q′ ₁ −q ₁).

Dividing both sides by \sqrt(p₁(1−p₁)) we get:

(p′ ₁ −p ₁)/\sqrt(p ₁(1−p ₁))=(x ₁₁ −p ₁ q ₁)/(\sqrt(p ₁(1−p ₁)q ₁(1−q₁))(q′ ₁ −q ₁)/\sqrt(q ₁(1−q ₁)).

Define r as the correlation coefficient between the combined allele G1and G2 and the combined causal alleles. By the definition of correlationcoefficient, the previous equation can be written as:

(p′ ₁ −p ₁)/\sqrt(p ₁(1−p ₁))=r(q′ ₁ −q ₁)/\sqrt(q ₁(1−q ₁)),

r=(p′ ₁ −p ₁)/\sqrt(p ₁(1−p ₁))\sqrt(q ₁(1−q ₁))(q′ ₁ −q ₁).

Given that q′₁−q₁<1−q₁,

r>(p′ ₁ −p ₁)/\sqrt(p ₁(1−p ₁))\sqrt(q ₁(1−q ₁))/(q′ ₁ −q ₁).

r ²>(p′ ₁ −p ₁)²/(p ₁(1−p ₁))q ₁/(1−q ₁).

In the NIH FSGS cohort, p₁=33% and p′₁=72%. If we assume that q₁>30%, weget an estimate r²>29%, that could be only explained by a short distancebetween the two variants.

Results

More than 50 genetic variants spanning the region including APOL1,without bias towards either gene were selected for fine mapping. Becausethe kidney disease risk allele(s) should have a high frequency inAfrican-Americans, as suggested by previous studies (Kopp et al., NatureGenet. 40:1175-1184, 2008; Kao et al., Nature Genet. 40:1185-1192,2008), causal alleles should be present in the sequence data of Africansavailable from the 1000 Genomes Project (available on the web at1000genomes.org). The data was searched for variants that were highlypolymorphic in Yoruba that were rare or absent in Europeans, asdisease-causing variants are expected to have this property. Inaddition, a single 6 bp deletion (rs71785313) in the coding region ofAPOL1 also identified by the 1000 Genomes Project that was observed inthree of the Yoruba samples was studied. Many of these variants have notbeen genotyped by the HapMap project.

An association analysis was performed with each of these variants anddisease, using DNA from 205 African-Americans with biopsy proven FSGSand no family history of FSGS and 180 African-American controls.Association between disease and each variant showed that the strongestassociations were all clustered in a 10 kb region centered on the lastexon of APOL1 (Table 3). These findings are summarized in FIG. 1A. Thestrongest association was obtained for the haplotype termed “G1”consisting of the two derived alleles for rs73885319 (S342G) andrs60910145 (I384M), in the last exon of APOL1. These two alleles werefound to be in perfect linkage disequilibrium (LD) (r²=1). The G1compound allele (342G:384M) had a frequency of 52% in the combined setof FSGS cases and 18% in controls (Table 4).

When logistic regression controlling for rs73885319 was performed, asecond strong signal was detected for a 6 bp deletion termed “G2”recently entered into dbSNP as rs71785313 (−/TTATAA; SEQ ID NO: 6) thatremoves amino acids N388 and Y389 (Table 5). Due to the extremely closeproximity of rs73885319, rs60910145, and rs71785313, the two alleles G1and G2 are mutually exclusive, as recombination between them is veryunlikely. FIG. 1B highlights variants which still showed statisticallysignificant associations. These results are in accordance with recentstudies (Freedman et al., Kidney Int. 75:736, 2009; Nelson et al., Hum.Mol. Genet. 19:1805-1815, 2010; Behar et al., Hum. Mol. Genet.19:1816-1827, 2010), which also identified multiple differentindependent signals of association. Allele G2 had a frequency of 23% inthe combined set of cases and 15% in the controls (Table 4).

Among the FSGS cases, all proven by kidney biopsy, 53 individuals wererecruited through the Brigham and Women's Hospital (BWH) from medicalcenters in the northeastern United States, and 152 individuals wererecruited in the US National Institutes of Health (NIH) FSGS GeneticStudy from 22 academic medical centers in the United States (MacKenzieet al., J. Am. Soc. Nephrol. 18:2987, 2007; Orloff et al., Physiol.Genom. 21:212, 2005). As controls, DNA from 180 individuals from the NIHBlood Bank and the National Cancer Institute-Frederick normal donorprograms were used.

Odds ratios for disease were computed using the NIH samples, as thesesamples were the best matched geographically. Table 6 shows the countfor each one of the six possible compound genotypes observable in eachcohort of cases and controls. By combining the two risk alleles G1 andG2, a χ² squared test showed no association with FSGS between sampleswith no risk alleles and one risk alleles (p=0.81). This supports acompletely recessive model. A second analysis comparing samples with oneor no risk alleles and samples with two risk alleles provided an oddsratio for FSGS of 10.5 (CI 6.0-18.4).

When comparing the number of samples with two risk alleles among the BWHcases and the NIH cases, as shown in Table 3, significant statisticaldifferences were observed among frequencies of alleles G1 and G2 using aFisher's exact test (p=0.04). This disparity cannot be explained by adifference in the amount of African ancestry, as presence of riskalleles implies African ancestry at the relevant locus, but may simplyreflect a difference in the frequency of allele G1 in the north easternUnited States.

Example 2 Replication in Hypertension-Attributed EKSD Methods

Selection criteria for controls and hypertension-attributed ESKD casesare described in detail in Freedman et al. (Kidney Int. 75:736, 2009).Briefly, self-reported African-Americans from North Carolina, SouthCarolina, Georgia, Virginia, or Tennessee were recruited.Hypertension-attributed ESKD cases were diagnosed with hypertensionprior to initiation of renal replacement therapy, and demonstratedhypertensive target end-organ damage (retinopathy or left ventricularhypertrophy) and low grade or absence of proteinuria. Only a minority ofcases had quantified urinary protein excretion. Patients with diabetic(type 1 and 2) ESKD were excluded, as were known cases of cystic kidneydisease, hereditary nephritis, and urologic causes of ESKD.

Geographically similar controls all denied a history of kidney diseaseand diabetes, or first-degree relatives with these diseases. Mostcontrols did not have direct measurements of arterial blood pressure orrenal function indices. Consequently, some controls may have had occultkidney disease, which would underestimate the effect size between casesand controls.

Results

Association of APOL1 variants and renal disease was tested in a muchlarger cohort of 1030 African-American cases with putative hypertensiveESKD and 1025 geographically matched African-American controls from WakeForest University. In this cohort 36 variants were investigated thatwere chosen based on strongest signals of positive selection in abroader region, nearby coding variants together with rs73885319 (G1) andrs71785313 (G2). The strongest association found was again forrs73885319 (p=1.1×10⁻³⁹, Table 7). Upon controlling for rs73885319, thestrongest association was again for rs71785313 (p=8.8×10⁻¹⁸, Table 8).Frequencies for these alleles are shown in Table 3.

With this larger population the mode of inheritance of G1 and G2 wasexplored. Cases and controls were partitioned into the six possiblegenotypes. One risk allele was associated with only a small increase inrenal disease risk (odds ratio 1.26, CI 1.01-1.56, p=0.047). Two riskalleles versus zero risk alleles yielded an odds ratio of 7.3 (CI5.6-9.5). Two risk alleles versus one risk allele gave an odds ratio of5.8 (CI 4.5-7.5). Overall, a recessive model best explains thesefindings and is in agreement with the analysis of FSGS samples.

Example 3 Evidence of Natural Selection Methods

Test for genetic divergence in African populations: To test forstatistically significant differentiation of allele frequency in betweentwo populations we assume that the difference in frequencies for a givenpolymorphism has mean 0 and variance cp(1−p), where p is the ancestralfrequency and c=2×F_(ST) (Ayodo et al., Am. J. Hum. Genet. 81:234-242.2007). Given the small size of the samples in the two populationsanalyzed, it is also important to model sampling noise, which hasvariance p(1−p)(1/N₁+N₂), where N₁ and N₂ are the total count for thealleles for the two populations. Therefore, to test for differentiationof frequency at a given allele, we model the difference as a normalrandom variable with mean 0 and variance p(1−p)(c+1/N₁+1/N₂) and wecompute for each allele a χ² statistic with 1 df.

Estimation of the age of the selected allele: Because of the presence ofa recombination hotspot in between APOL1 and MYH9 (Frazer et al., Nature449:851-861, 2007), SNP rs11912763, the variant most correlated with G1available in Hapmap, has genetic distance of about 0.2 centimorgans fromAPOL1 cSNPs rs73885319 and rs60910145, despite a physical distance ofless than 25 kb from APOL1. The derived allele for SNP rs11912763,absent outside of Africa, has a prevalence of about p=73% in haplotypescontaining the G1 allele. If we assume that the G1 allele arose in ahaplotype already containing the rs11912763 derived allele, then theprevalence of the derived allele for rs11912763 in haplotypes containingthe G1 allele could not have decreased at a rate faster than theexpected frequency of recombination 1−c per generation. This leads to anestimate for the number t of generations

(1−c)^(t) ≤p,

t≥log(p)/log(1−c),

from which we obtain a lower bound of about 150 generations, using thevalues for c and p as above. If we assume an average of 20 years pergeneration, this estimate suggests an age of at least 3,000 years forallele G1. Given the prevalence of about p=72% for the rs2239786 derivedallele in haplotypes containing the G2 allele, a similar estimate alsoholds for the age of allele G2.

Results

The chromosomal region where APOL1 resides has previously been shown tobe a candidate for positive selection in the Yoruba population using thelong-range haplotype method (LRH) (Frazer et al., Nature 449:851-861,2007), the integrated haplotype score (iHS) (Voight et al., PLoS Biol.4:446, 2006; Barreiro et al., Nature Genet. 40:340-345, 2008), the rMHH(Kimura et al., PLoS One 2:e286, 2007), and the composite of multiplesignals (CMS) (Grossman et al., Science 327:883, 2010). The G1 and G2allele was present in all the Yoruba Hapmap samples and the extendedhaplotype homozygosity (EHH) (Sabeti et al., Nature 419:832-837, 2002)was computed for each one of the three alleles after phasing the datausing Beagle (Browning and Browning, Am. J. Hum. Genet. 84:210-223,2009) (FIG. 2). The iHS score was not computed, as the proximity of arecombination hotspot makes this particular computation unstable.

The frequency of allele G1 was also compared in Yoruba samples fromNigeria and Luhya samples from Kenya to verify statistically significantdifferences in these two populations. The Yoruba population from Nigeria(YRI) and the Luhya population from Kenya (LWK), despite beingrespectively from West Africa and East Africa, are very closely relatedgenetically with F_(ST)=0.0043. To test for selection, a model ofallele-frequency differentiation between two populations was used (Ayodoet al., Am. J. Hum. Genet. 81:234-242. 2007), which corrects for geneticdrift. The results showed that differentiation for rs73885319, whosefrequencies are 38% in the Yoruba and 5% in the Luhya, is highlysignificant (F_(ST)=0.16 and p=3.53×10⁻⁹). Interestingly, variantrs73885319 was the second most highly differentiated variant in thesetwo populations across the whole genome. The frequencies of variantrs71785313, respectively 0.08 and 0.07, did not show any significantdifferentiation. Results of this analysis for the region in between34,900 kb and 35,100 kb (NCBI 36) are shown in Table 9.

By analyzing the pattern of linkage disequilibrium between these SNPs,it appears likely that alleles G1 and G2 are at least 3,000 years old.The true age is likely older than this number, but not by orders ofmagnitude, and it might coincide with the Bantu expansion event, aseries of migrations across sub-Saharan Africa that is estimated to havetaken place between 4,500 and 5,000 years ago (Excoffier et al., Am. J.Phys. Anthropol. 30:151-194, 2005). In particular, frequencydifferentiation of allele G1 between two populations from West and EastAfrica points to natural selection having acted after the Bantuexpansion, either to raise the frequency in Yoruba or to decrease thefrequency in Luhya.

Example 4 APOL1 and Resistance Against Trypanosome Methods

Expression of ApoL1 proteins: Two independent systems were used forexpression of recombinant ApoL1 in Escherichia coli and in 293T cells.The various ApoL1 mutants were generated by site-directed mutagenesisand expressed in E. coli essentially as described in Lecordier et al.(PLoS Pathog. 5:e1000685, 2009), except that the pStaby1.2 plasmid(Delphi Genetics, Gosselies, Belgium) was used. For production of ApoL1protein in 293T cells with and without the G1 and G2 risk mutations, animage clone containing the ApoL1 cDNA lacking the G1 and G2 mutations(reference sequence BC141823) was purchased from Open Biosystems(Huntsville, Ala.). This cDNA was provided in the pCMV-SPORT6 expressionvector. The G1 and G2 mutations were introduced by synthesis of cDNAminigene fragments (Integrated DNA Technologies, Coralville, Iowa)containing the corresponding mutations flanked with 5′ AleI and 3′ XbaIrestriction sequences. The minigene fragments were then cloned into theparental vector replacing the sequence between the AleI and XbaIrestriction sites. The resultant vectors were used to transfect 293Tcells using Fugene (Promega, Madison, Wis.). The transfection media wasreplaced with OPTI-MEM reduced serum media without phenol red (LifeTechnologies/Invitrogen, Carlsbad, Calif.) at 12 hours posttransfection. At 72 hours post transfection the supernatants wereharvested and concentrated 100 fold using an Amicon Ultracel—10Kcentrifugal filter unit (Millipore, Billerica, Mass.). The media wasexchanged by centrifugation within the Ultracel filters with freshIscove's Modified Dulbecco's Medium for compatibility with thetrypanosome killing assay.

Trypanolytic assays: The evaluation of trypanolytic activity of thevarious ApoL1 mutants was performed as described in Lecordier et al.(PLoS Pathog. 5:e1000685, 2009).

Results

ApoL1 is the trypanolytic factor of human serum (Vanhamme et al., Nature422:83-87, 2003; Perez-Morga et al., Science 309:469, 2005) and confersresistance to the Trypanosoma brucei brucei parasite. T b. brucei hasevolved into two forms, Trypanosoma brucei gambiense and Trypanosomabrucei rhodesiense (Gibson Parasitol. Today 9:255-257, 1986; GibsonTrends Parasitol. 18:486-490, 2002) which have both acquired the abilityto infect humans. FIGS. 3A and B show the relative distribution ofinfections by T b. rhodesiense and T b. gambiense. Since these parasitesexist only in sub-Saharan Africa, it is plausible that the APOL1 genehad undergone natural selective pressure to counteract the trypanosomeadaptations.

T b. rhodesiense can grow in humans because of a serumresistance-associated (SRA) protein that interacts with the C-terminalhelix of ApoL1 and inhibits its anti-trypanosomal activity (Xong et al.,Cell 6:839-846, 1998; Vanhamme et al., Nature 422:83-87, 2003). A recentstudy showed that mutations and deletions engineered into this helixprevent SRA from binding to ApoL1 (Lecordier et al., PLoS Pathog.5:e1000685, 2009). The 6 bp deletion rs71785313 defining the G2 alleleis located exactly at the SRA binding site in the ApoL1 C-terminalhelix.

Analysis of the in vitro lytic potential of 77 human plasma samples wasconducted on T b. brucei, T b. rhodesiense, and T b. gambiense. Whileall samples efficiently lysed T b. brucei, none lysed T b. gambiense and46 lysed normal human serum (NHS)-resistant T b. rhodesiense clones. AllT b. rhodesiense lytic samples belonged to G1, G2 or both genotypes. Asmeasured by titration upon serial dilution, the lytic potential of theseplasmas against NETS-resistant (SRA+) T b. rhodesiense was higher for G2than for G1, whereas it was similar for both genotypes againstNHS-sensitive (SRA−) parasites (FIG. 4A). While lysis of T b.rhodesiense by G2 could be explained by the incapacity of this mutantApoL1 to bind SRA, this conclusion did not hold for G1 plasmas, whereApoL1 still efficiently bound to SRA (FIG. 4B).

These results were confirmed with recombinant ApoL1 proteins. TheS342G/I384M (G1) and delN388/Y389 (G2) variants killed bothNHS-sensitive (SRA−) and NHS-resistant (SRA+) T b. rhodesiense parasites(FIG. 4C), but not T b. gambiense. While G2 was more active than G1 onNHS-resistant T b. rhodesiense, the reverse was true on NHS-sensitiveparasites. ApoL1 variants with either S342G or I384M alone were lesslytic against T b. rhodesiense than was the combination of the twomutations, whereas the S342G/I384M/delN388/Y389 variant was not moreactive than delN388/Y389 alone (FIG. 4C). As shown in FIGS. 4D and E,all measured features of the T b. rhodesiense lysis process (kinetics,transient inhibition by chloroquine, typical swelling of the lysosome)were similar to those observed on T b. brucei with either NHS orrecombinant ApoL1 (Perez-Morga et al., Science 309:469-472, 2005).Therefore, deletion of N388/Y389 was necessary and sufficient to preventinteraction with SRA and to allow ApoL1 to kill T b. rhodesiense,whereas the combination of S342G and I384M was required for maximalability to kill T b. rhodesiense despite the binding of SRA. None ofthese mutations affected the resistance of T b. gambiense.

Example 5 Predictive Power of APOL1 SNPs

HIV negative individuals carrying one APOL1 risk allele at rs73885319and one APOL1 risk allele at rs71785313 have a predicted 4.3 foldincrease in risk of FSGS over the (African American) population average.40% of these individuals have a predicted 1.6 fold increased, while theremaining 60% have a predicted 5.6 fold predicted risk; the individualsreceiving an exaggerated prediction of risk represent 22 out of 1000individuals tested. Similar although smaller improvements in riskestimates occur for other APOL1 risk strata.

The ROC C statistic was calculated for FSGS. For FSGS, the C statisticfor at least one APOL1 risk allele was 0.822. In HIV positiveindividuals, the C statistic for FSGS for at least one APOL1 risk allelewas 0.865. This increase in the C statistic represents a 3% reduction inresidual ignorance of FSGS risk.

Example 6 Nucleic Acid-Based Analysis of Genetic Predisposition to RenalDisease

The methods disclosed herein are used for evaluating if a subject has oris at risk for developing renal disease. For example, the methods can beused to determine if a subject is at risk for FSGS, or is at risk forhypertensive ESKD. One skilled in the art will appreciate that methodsthat deviate from these specific methods can also be used tosuccessfully determine if a subject is at risk for renal disease.

In one example, a sample including nucleic acids can be obtained from asubject who is suspected to have a genetic predisposition to renaldisease, such as FSGS or hypertensive ESKD. The subject can have familymembers who have had FSGS or hypertensive renal disease. In anotherexample, a sample including nucleic acids can be obtained from a subjectthat is of African ancestry. In a further example, a sample includingnucleic acids is obtained from a subject with African (such asAfrican-American) ancestry who is infected with HIV.

In a further example, a sample including nucleic acids is obtained froma subject who has renal disease, wherein it is of interest to determineif the subject has hypertensive ESKD. For example, a sample can beobtained from a subject who presents with a reduced glomerularfiltration rate (GFR) or other laboratory evidence of renal impairment(such as elevated blood urea nitrogen (BUN) or abnormal renalhistology), or someone with the clinical presentation (symptoms) ofrenal disease, such as fatigue and liquid retention. Additionalindicators of renal disease that can suggest chronic renal failureinclude hyperkalemia, acidemia, elevated serum creatinine levels and/orthe uremic syndrome. A renal biopsy can be obtained from the subject todetermine if the subject has FSGS or hypertensive nephrosclerosis.

In some particular embodiments of the method, the subject isseropositive for the HIV virus, and the test is performed to predictwhether the subject is likely to develop renal disease, such as chronicrenal failure, such as renal failure caused by FSGS. In otherembodiments, the subject is someone who has clinical and laboratoryevidence of early renal disease and the genetic test is performed toconfirm the diagnosis of renal disease. For example, the subject may bean African-American with clinical evidence of early renal failurewithout a known etiology. Alternatively, the subject may have had arenal biopsy performed with inconclusive or ambiguous results. In theseinstances, the genetic test is performed to arrive at a diagnosis ofchronic renal disease (or FSGS) with a higher degree of clinicalcertainty than would otherwise be possible. The genetic test can be usedin association with other clinical signs and symptoms to assign adiagnosis, and from the diagnosis greater prognostic certainty can beprovided to the subject. Alternatively, the genetic test can be used toprovide a more specific diagnosis or etiology for chronic renal failure,as may be needed in research studies or for the selection of anappropriate therapeutic regimen.

In some examples a sample including nucleic acids is obtained from asubject with lupus nephritis or sickle cell anemia. These subjects canbe tested to determine their haplotype at the time of diagnosis. Inother examples a sample including nucleic acids is obtained from asubject with diabetes mellitus (type 1 or type 2), IgA nephropathy,and/or renal vasculitis.

The finding of a susceptibility haplotype can initiate screeningannually or biannually for protein, using albumin/creatinine ratio, suchas beginning at about age 12 or about age 15. For example, subjects whoare found to have a condition that is associated with renal injury,including prematurity, small birth weight, obesity, hypertension,systemic lupus erythematosus, sickle cell anemia, diabetes mellitus, andHIV-1 infection can be screened using the methods disclosed herein.

To perform the method, a biological sample of the subject is assayed.The sample can, for example, be a blood sample or a buccal sample.Methods of isolating nucleic acid molecules from a biological sample areroutine, for example using a commercially available kit to isolate DNA.Nucleic acid molecules isolated from PBMCs or any other biologicalsample can be amplified (for example, by PCR) using routine methods toform nucleic acid amplification products.

It is determined if the individual has an APOL1 SNP (such as a G atrs73885319, a G at rs60910145, and/or a 6 base pair deletion atrs71785313) using standard methods, such as real-time PCR (for example,a TAQMAN® assay), allele-specific PCR, or sequence analysis. Thepresence of at least one APOL1 SNP indicates that the subject is at riskfor developing renal disease. For example, the methods can be used todetermine if a subject is at risk for FSGS, or is at risk forhypertensive ESKD.

Thus, in some cases, it is determined if the individual has an APOL1 SNP(such as a G at rs73885319, a G at rs60910145, and/or a 6 base pairdeletion at rs71785313) using standard methods, such as real-time PCR(for example, a TAQMAN® assay), allele-specific PCR, or sequenceanalysis. The presence of at least one APOL1 SNP indicates that thesubject is at risk for developing renal disease. For example, themethods can be used to determine if a subject is at risk for FSGS, or isat risk for hypertensive ESKD.

In another embodiment, the methods can be used to identify protectivealleles in a subject that are associated with the absence of renaldisease. In this instance, the detection of protective alleles in abiological sample may be indicative of a lower risk for developing renaldisease in the subject.

Example 7 Nucleic Acid-Based Analysis of Resistance to Trypanosoma

The methods disclosed herein are used for evaluating if a subject has aresistance to disease associated with Trypanosoma infection. Forexample, the methods can be used to determine if a subject hasresistance to African trypanosomiasis (sleeping sickness) caused by T.brucei. One skilled in the art will appreciate that methods that deviatefrom these specific methods can also be used to successfully determineif a subject has a resistance to disease associated with Trypanosomainfection.

In one example, a sample including nucleic acids can be obtained from asubject who is suspected to be at risk for disease associated withTrypanosoma infection. The subject can live in, have traveled to, orplan to travel to an area where Trypanosoma parasites are endemic, forexample, sub-Saharan Africa.

To perform the method, a biological sample of the subject is assayed.The sample can, for example, be a blood sample or a buccal sample.Methods of isolating nucleic acid molecules from a biological sample areroutine, for example using a commercially available kit to isolate DNA.Nucleic acid molecules isolated from PBMCs or any other biologicalsample can be amplified (for example, by PCR) using routine methods toform nucleic acid amplification products.

It is determined if the individual has an APOL1 SNP (such as a G atrs73885319, a G at rs60910145, and/or a 6 base pair deletion atrs71785313) using standard methods, such as real-time PCR (for example,a TAQMAN® assay), allele-specific PCR, or sequence analysis. Thepresence of at least one APOL1 SNP indicates that the subject has aresistance to disease associated with Trypanosoma infection. Forexample, the methods can be used to determine if a subject is hasresistance to disease associated with infection with T brucei.

In another embodiment, the methods can be used to identify APOL1 SNPs(such as an A at rs73885319, a T at rs60910145, and absence of a 6 basepair deletion at rs71785313) in a subject that are associated withdecreased resistance or susceptibility to disease associated withTrypanosoma infection. In this instance, the detection of these SNPs ina biological sample may be indicative of decreased resistance orincreased susceptibility of the subject to disease associated withTrypanosoma infection.

Example 8 Genetic Variation in APOL1 and Age at Hemodialysis Initiationin African Americans

African Americans have a markedly higher incidence of end-stage renaldisease (ESRD) compared with other racial groups. Two coding sequencerisk alleles in the APOL1 gene, found only in individuals of recentAfrican ancestry, have been identified as risk alleles for renal diseasein African Americans. We tested whether these risk alleles were alsolinked to age of initiation of chronic hemodialysis.

Methods: We performed a cross-sectional study of 407 non-diabeticAfrican-Americans with ESRD who participated in Accelerated Mortality onRenal Replacement (ArMORR), a prospective cohort study of incidentchronic hemodialysis subjects from across the United States. We examinedage of initiation of chronic hemodialysis according to APOL1 riskalleles (G1 and G2). Analysis of variance was used to compare mean ageat dialysis initiation, and multivariate linear regression modeling wasused to adjust for potential confounders.

Results: African American subjects carrying two copies of the G1 riskallele initiated chronic hemodialysis at a mean age of 49.0±14.9 years,significantly earlier than subjects with one copy of the G1 allele(55.9±16.7 years: p=0.014) or those without any risk allele (61.8±17.1years; p=6.2×10⁻⁷). The G1 relationships remained statisticallysignificant in multivariate analysis adjusting for socio-demographic andother potential confounders. G2 risk allele was not linked to age ofchronic hemodialysis initiation; however, limited sample size in thisanalysis precluded definitive conclusions.

Conclusion: Genetic variations in the APOL1 gene identify AfricanAmericans that initiate chronic hemodialysis at an earlier age. Earlyinterventions to prevent progression of kidney disease may benefit thishigh-risk population.

INTRODUCTION

African Americans have a four-fold greater risk of end stage renaldisease (ESRD) compared with white Americans (Klag et al., JAMA277:1293-1298, 1997; System, N.I.o.D.a.D.a.K.D. National Institutes ofHealth, Editorial, 2010). In 2009, the mean age for African Americans atthe start of renal replacement treatment was 59.2 years, compared with66.8 years in Caucasians (System, supra). This may be due in part to anaccelerated progression of renal disease in African Americans (Hsu etal., J Am. Soc. Nephrol. 14:2902-2907, 2003; Walker et al., JAMA268:3085-3091, 1992; Derose et al., Kidney Int. 76:329-637). Severalstudies have found that the high prevalence of ESRD in African Americanscannot be fully explained by socioeconomic differences or differences inaccess to medical care (Klag et al., supra; Tarver-Carr et al., J Am.Soc. Nephrol. 13:2363-2370, 2002). Thus, it is thought that biologicfactors, such as genetic differences, contribute to this disparity.Indeed, previous studies have demonstrated strong familial aggregationof kidney disease in African Americans (Freedman et al., J Am. Soc.Nephrol. 8:1942-1945, 1997). Two recent studies used genetic admixturemapping to identify a region of chromosome 22 that explained theincreased kidney disease risk in African Americans (Kao et al., Nat.Genet. 40:1185-1192, 2008; Genovese et al., Science 329:841-845, 2010).

Genovese et al. identified sequence variants in apolipoprotein L-1(APOL1) as risk alleles for focal segmental glomerulosclerosis (FSGS)and hypertension-attributed ESRD (H-ESKD) in African Americans (Genoveseet al., Science, supra; Genovese et al., Kidney Int. 78:698-704, 2010).APOL1 is located adjacent to the MYH9 gene on chromosome 22, a locusthat has previously been reported to explain the high risk of renaldisease in African Americans (Kao et al., supra). Interestingly, APOL1risk proteins have lytic activity against a subspecies of trypanosomesknown to cause African sleeping sickness. Carrier status may haveprovided a selective evolutionary advantage and thus maintained theserisk alleles in the African population. The two risk alleles found toconfer an elevated risk for FSGS and H-ESRD includes “G1,” a two locusallele found in a 10-kb region in the last exon of APOL1, and “G2,” asix base pair deletion located in close proximity to the G1 risk allele(Genovese et al., Science 329:841-845, 2010). These risk alleles inAPOL1 are only found in individuals of African descent with allelefrequencies of 38% for G1 and 8% for G2 in the African Yorubapopulation. These alleles appear to act in a recessive manner, with a 7to 10 fold increased risk of H-ESKD or FSGS conferred by the presence ofa risk-associated allele in both copies of APOL1.

Given the association between APOL1 risk alleles and non-diabetic renaldisease in African Americans, we tested whether African Americans withESRD and who are homozygous for APOL1 risk alleles progress to ESRD atan earlier age than those who do not have these risk alleles. The testswere performed in a cohort of non-diabetic African American subjectsinitiating chronic hemodialysis in the United States.

Results

Subject characteristics, including demographic information, income,vascular access, cause of ESRD and laboratory values are summarized inTable 10. The mean age of hemodialysis initiation among all subjects was55.2±17.1 years.

When subjects were stratified into six unique groups according to thenumber of G1 or G2 risk alleles, only subjects with two G1 risk alleleshad significantly lower mean age at hemodialysis initiation compared tosubjects without these APOL1 risk alleles (Wt+Wt=61.8±17.0 vs.Wt+G1=55.9±16.7 [p=0.152]; vs. G1+G1=49.0±14.9 [p=3.0×10⁻⁶]; vs.G1+G2=49.3±17.0 [p=1.83×10⁻⁴], FIG. 5). In contrast, subjects with oneor two G2 risk alleles, but no G1 risk alleles, did not beginhemodialysis at an earlier age compared with subjects who lacked the G1or G2 risk alleles (Wt+G2=58.1±16.3 [p=0.96], G2+G2=48.9±15.3 [p=0.09]).However, the number of individuals with the G2+G2 risk alleles wassmall. We therefore conducted subsequent analyses only with G1 riskalleles. Decreased power from exclusion of subjects with G2 risk allelesdid not dramatically change the results of the analysis. Including allsubjects, regardless of G2 risk, mean age at hemodialysis wassignificantly lower among those with two G1 risk alleles compared tothose without G1 risk alleles (p=1.0×10⁻⁶).

Table 11 shows subject characteristics according to whether subjects hadzero, one, or two copies of the G1 risk allele. The p-values in Table 11represent comparisons between the genotypes. G1 homozygotes,heterozygotes, and subjects without the G1 risk allele had a similarproportion of male subjects and similar income distribution, and similarlocations of hemodialysis initiation. Subjects with and without G1alleles had similar systolic and diastolic blood pressures, parathyroidhormone levels, calcium levels, hemoglobin concentration, and albuminconcentrations. Subjects with G1 risk alleles tended to have higherserum creatinine levels (p=1.0×10⁻⁶), perhaps due to their age. Toinvestigate possible confounders between G1 risk allele and age athemodialysis initiation, we also investigated factors independentlycorrelated with mean age at hemodialysis initiation; BMI (r=−0.224,p=1.2×10⁻⁴); serum creatinine (r=−0.439, p=1.5×10⁻¹⁴); systolic bloodpressure (r=−0.188, p=1.3×10⁻³); diastolic blood pressure (r=−0.490,p=9.2×10⁻¹⁹). We estimated eGFR levels using the MDRD formula and foundthat subjects with G1 risk alleles had lower eGFR levels at ESRDinitiation (p=8.1×10⁻⁵).

In consonance with other U.S. renal study populations, in nearlythree-fourths of our subjects ESRD was caused by hypertension. Othercauses of ESRD in our population, including HIV, inflammation, toxins,etc. were grouped together as ‘other.’ In stratified analyses by causeof ESRD, the mean age at initiation of hemodialysis remained younger inH-ESRD subjects with G1 risk alleles but not in subjects with otherreported causes of ESRD, FIG. 6.

Subjects with ESRD due to causes other than hypertension initiatedchronic hemodialysis at an earlier mean age than subjects with H-ESRD(50.6±18.0 years vs. 58.1±16.3 years, p=7.8×10⁻⁴). In multivariateregression models, the presence of G1 risk alleles remainedsignificantly associated with early hemodialysis initiation afteradjustment for demographic and socioeconomic variables and cause of ESRDamong non-diabetics (Table 12). The average values in Table 12 representthe predicted values estimated from multivariate regression equationscontrolling for socio-demographic and clinical characteristics. In asimilar model, when we did not stratify by cause of ESRD (hypertensionvs other), we found that the G1 allele remained associated with age ofhemodialysis initiation.

Discussion

We aimed to determine if the G1 or G2 sequence risk alleles of the APOL1gene (which are associated with an increased risk of renal disease inAfrican Americans) are associated with initiating chronic hemodialysisat a younger age—a marker of the severity of progressive CKD. We foundthat African Americans with two copies of the G1 risk allele initiatedchronic hemodialysis approximately ten years earlier than those withoutthis allele. Subjects with one copy of the G1 allele initiatedhemodialysis an average of six years earlier. These estimates wereunchanged after adjustment for a variety of socio-demographic factors.The presence of the G2 allele may be associated with the initiation ofhemodialysis at a younger age.

APOL1 risk alleles are associated with FSGS and H-ESKD (Genovese et al.,Kidney Int., supra). This study shows that these risk alleles are alsoassociated with age at first start of chronic hemodialysis, a measure weused as a surrogate for age of developing ESRD. Our data support theconceptual model that APOL1 risk alleles either trigger onset of renaldisease at an earlier age, or once initiated, alter the rate ofprogression.

Several factors may influence increased risk for ESRD in AfricanAmericans. Milder forms of kidney disease, CKD stages are less prevalentin African Americans while stage IV CKD and ESRD are much more common(System, supra; Hsu et al., supra; Volkova et al., J. Am. Soc. Nephrol.19:356-364, 2008). Consistent with this, in the ArMORR study, theoverall mean age at hemodialysis initiation for Caucasians is 65.5±14.9years, compared to 57.8±15.4 years in African Americans (p<1.0×10⁻⁴)(Shurraw et al., Am. J. Kidney Dis. 55:875-884, 2010). This isconsistent with an accelerated progression of ESRD in these subjects.This could also be explained by an alternative pathogenesis for ESRD insome African Americans, leading to early clinical onset of ESRD,potentially mediated by mutations in the APOL1 gene. Several factorshave been associated with the rate of progression to ESRD in AfricanAmericans, including, e.g., proteinuria, 25-hydroxyvitamin D levels, andlate referral to specialty care. One or more of these risk factors maymediate the relationship between APOL1 risk alleles and ESRD in thispopulation (Melamed et al., J. Am. Soc. Nephrol. 20:2631-2639, 2009). Inthis study we found that body mass index and income were unlikely toconfound the association between APOL1 genetic risk alleles and earlierage at hemodialysis initiation.

Subjects with two copies of the G2 risk allele may initiate hemodialysisat a younger age, while subjects with one copy of the G2 risk alleleappear to initiate hemodialysis at approximately the same age assubjects without any risk alleles.

As there was less power to detect effects of the rarer G2 risk allele,it is less clear to what extent this characteristic affects age athemodialysis initiation. Post-hoc power analysis revealed a sample sizeof 34 per G2 risk allele group is required to obtain a power of 0.8, and45 to obtain a power of 0.9. Based on our current sample sizes it islikely that homozygous G2 risk allele groups were underpowered to findany significant differences between age of hemodialysis initiation.Also, just one copy of the G1 allele was associated with a younger ageat hemodialysis initiation.

In conclusion, genetic variation in APOL1 is associated with earlieronset of ESRD in African Americans without diabetes mellitus as theetiology of end stage renal failure, and thus APOL1 genetic screeningcan be used to identify patients at risk so that preventativeinterventions can be initiated much earlier than is currently practiced.

Methods

Subjects

Subjects were African Americans with non-diabetic ESRD enrolled inAccelerated Mortality on Renal Replacement (ArMORR), a prospectivecohort study of 10,044 subjects who initiated chronic hemodialysis atany of 1056 US hemodialysis centers operated by Fresenius Medical Care,North America between June 2004 and August 2005.[15] The study wasapproved by the Institutional Review Board (IRB) of the MassachusettsGeneral Hospital (MGH), which waived the need for informed consent ofthis repository. Blood samples from 407 non-diabetic African Americanswere available for DNA extraction.

Data Collection

Data were collected prospectively by care-givers and includeddemographics, body mass index, co-morbidities, hemodialysis access(catheter, graft, or fistula), and reported cause of ESRD. Laboratorytests on blood samples collected within 14 days of hemodialysisinitiation were performed by a central laboratory (Spectra East,Rockland, N.J.) and included albumin, creatinine, calcium, phosphate,and hemoglobin, measured using standard multisample automated analyzers.Intact parathyroid hormone (PTH) was measured using Nichols Bio-intactassay of full-length 1-84 PTH. Subject's eGFR levels were estimatedusing the Modification of Diet in Renal Disease (MDRD) formula (Levey etal., Ann. Intern. Med. 130:461-470, 1999).

Each subject received chronic hemodialysis at an outpatient FreseniusMedical Center North American facility. Because age of initiation ofchronic hemodialysis may differ by region and income, median householdincome of African Americans living in the zip code for each facility wasdetermined from U.S. Census data for the year 2000 and used as anestimate of socioeconomic status. (Kinchen et al., Ann. Intern. Med.137:479-486, 2002.) We divided subjects into three equally sized groups(high, medium, and low socioeconomic status) based on median householdincome of African Americans in the zip code in which they initiateddialysis. We also assigned subjects to one of three geographic regionsbased on their hemodialysis facility's zip code and U.S. Census Regions(U.S. Census, available from:http://factfinder.census.gov/servlet/DTTable?_bm=y&-context=dt&-ds_name=DEC_2000_SF3_U&-CONTEXT=dt&-mt_name=DEC_2000_SF3_U_P151B&-tree_id=403&-redoLog=false&-all_geo_types=N&-geo_id=01000US&-geo_id=02000US1&-geo_id=02000US2&-geo_id=02000US3&-geo_id=02000US4&-search_results=01000US&-format=&-_lang=en,retrieved 2002). These regions included Northeast (Connecticut,Massachusetts, Maine, New Hampshire, New Jersey, Pennsylvania, PuertoRico, Rhode Island), Midwest/West (Arizona, Colorado, Illinois, Kansas,New Mexico, Missouri, Minnesota, Montana, Ohio, Wisconsin), and South(Alabama, Arkansas, Washington D.C., Delaware, Florida, Georgia,Kentucky, Louisiana, Maryland, Mississippi, North Carolina, Oklahoma,South Carolina, Tennessee, Texas, Virginia, and West Virginia). PuertoRico and the Island Areas are not part of any census region or censusdivision. For this reason, Puerto Rico was assigned to the North Eastregion based on Standard Federal Regions where Puerto Rico is grouped inRegion III with New York and New Jersey (Federal Regions, availablefrom:http://www.atsdr.cdc.gov/WebMaps/helpcontent/MapOptionsAdvance.asp#regions,retrieved 2010).

Genotyping

Genomic DNA was extracted from whole blood stored in PaxGene tubes usinga protocol adapted from PreAnalytix using a QiagenAutoPure extractionrobot (Harvard Partners Center for Genetics and Genomics, Cambridge,Mass., USA). In all samples, DNA quality was assessed with 260/280 ODratios. The patients' DNA samples were diluted in water to 10 nanogramsper microliter, and 300 nanograms of each DNA sample were sent toPolymorphic DNA Technologies in Alameda, Calif., which provided assaydesign, oligonucleotide primers, PCR amplification, DNA sequencing anddata analysis. Polymorphic DNA Technologies uses Sanger dideoxy DNAsequencing and employs automated high throughput capillaryelectrophoresis DNA sequencing instruments and is less prone to errorrates, especially when two alleles, such as G1 and G2, are juxtaposedclose to each other.

We considered both G1 and G2 risk alleles and classified the subjects byAPOL1 risk allele status into groups depending on the number of G1 or G2alleles present. This created six unique groups (WT+WT, G1+WT, G1+G1,G2+WT, G2+G2, G1+G2). Due to mutual exclusivity of G1 and G2, nosubjects had more than two risk alleles in total (FIG. 5). We thenconsidered G1 and G2 risk alleles separately and compared the age ofhemodialysis initiation in subjects with zero, one, or two copies ofeach allele.

Statistical Analysis

Analysis of variance (ANOVA) with Sidak post-hoc tests was used tocompare mean ages at hemodialysis initiation and other relevantcontinuous variables across genotypic groups. Pearson correlationcoefficients were used to examine the associations between continuousvariables. Chi-squared tests were used for categorical variables.Multivariate linear regression modeling was used to obtain the averagepredicted age of hemodialysis initiation for G1 risk alleles, excludingG2 risk alleles, after adjustment for socioeconomic, demographic andclinical factors. All statistical analyses were performed using SASversion 9.2 software (Cary, N.C.) and STATA version 11 (College Station,Tex.). Two-tailed p-values of <0.05 were considered significant.

Example 9 An APOL1 Gene Inversion as a Risk/Resistance Allele

As discussed above, the G1 and G2 variants are located in the C-terminalend of the APOL1 gene product. APOL1, APOL2, and APOL4 are located inclose proximity to one another on chromosome 22 (see FIG. 7A, not toscale).

The tail to tail (5′ to 5′) arrangement of APOL1 and APOL2 suggestscoordinated regulation, a prediction that has been confirmed with HapMapgene expression data (see FIG. 8).

Using bioinformatics, we discovered another APOL1 gene risk/resistanceallele that is a chromosomal rearrangement. The chromosomalrearrangement inversion is predicted to invert a segment of DNAincluding the 5′ end of APOL4, all of APOL2, and the 5′ end of APOL1(see FIG. 7B). Individuals with the rearrangement inversion on a givenchromosome have several important changes:

a) The reference APOL4 gene is replaced by an APOL1/APOL4 hybrid gene;

b) The reference APOL1 gene is replaced by an APOL4/APOL1 hybrid gene;

c) APOL4 expression is driven by the reference APOL1 promoter, and APOL1is driven by the reference APOL4 promoter; and

d) APOL1 and APOL2 coordinated expression may now be unlinked.

We validated the existence of the inversion at the APOL1/APOL4 junctionusing PCR in human samples (see FIG. 9).

The inversion in the APOL1 gene may result in replacement of up to threeexons in APOL1 by sequence from APOL4 (e.g., the inversion may result inreplacement of all or a portion of only the first exon, all or a portionof the first and/or second exon, and/or all or a portion of the first,second, or third exon of the APOL1 gene). These three exons may cover arange of 2000-2500 base pairs of genomic DNA (e.g., in a range of fromabout 100 base pairs to about 3000 base pairs of genomic DNA, such as arange from 1000 base pairs to about 2500 base pairs of genomic DNA), andmay encode a maximum of about 420 base pairs of transcript (e.g., arange of from about 20 base pairs to about 500 base pairs of transcript,such as from about 100 base pairs to about 420 base pairs of transcriptDNA). The actual coding sequence replaced in the APOL1 protein may onlycode for 1 to about 30 amino acids, e.g., about 10 to about 20 aminoacids, e.g., about 14 amino acids from APOL4. These substituted aminoacids may all appear in the preprotein portion of the hybrid APOL4/APOL1protein and all or a portion of the replaced amino acids may be cleaveddepending upon the extent and actual sequence of the inversion.

As shown in FIG. 10, the sequence in the inverted chromosome isreference APOL1 at the 5′ end and reference APOL4 at the 3′ end. Thebreakpoints on hg18 are at approximately chr22:34,981,580 tochr22:34,981,980 at the APOL1 end, and chr22:34,927,460 tochr22:34,927,060 at the APOL4 end.

The functional consequences of the inversion are predicted to be one ormore of the following:

a) the native APOL1 promoter is eliminated, and replaced by a promoterthat either expresses APOL1 at much lower levels or may not expressAPOL1 at all;

b) in individuals with the inversion and another SNP, rs9610445 (the Callele), an essential splice site is eliminated, causing an alterationin the transcript with potential functional consequences (see FIG. 11A);

c) in individuals with the inversion and another SNP, rs6000181 T(minor) allele, a methionine start site is eliminated, potentiallyaltering translation of APOL1 (see FIG. 11B).

d) APOL1 and APOL2 expression are no longer coordinated; and/or

e) The N-termini of ApoL1 and ApoL4 proteins are exchanged. Under normalconditions, this may have no effect, as APOL1 has a signal peptide thatis cleaved prior to export from the cell, effectively removing the aminoacids contributed by APOL4. However, in the setting of dramatic APOL1upregulation that we have observed when cells are exposed toinflammatory factors, the APOL4-encoded region may not be efficientlycleaved and could affect molecular function.

Despite the unusually large odds ratio for renal disease associated with2 APOL1 renal risk variants (G1 and G2), some individuals with 2 riskvariants do not develop disease, while some with 0 or 1 variant dodevelop disease. The functional properties of the inversion may be a“G3” that will improve predictive value of APOL1 testing. Thus, theidentification of the G1, G2, and/or G3 risk alleles in a human subjectis predictive of a genetic predisposition to and/or increased risk ofthe development of renal disease in the human subject.

In addition, for the reasons given herein, G3 may also be a resistanceallele that can be detected alone or in combination with G1 and/or G3 ina human subject to determine a resistance to a disease associated withinfection by a Trypanosoma spp. in the human subject.

Other Embodiments

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features hereinbefore set forth.

All publications and patent applications mentioned in thisspecification, including the priority application, U.S. Application Ser.No. 61/325,343, are herein incorporated by reference to the same extentas if each independent publication or patent application wasspecifically and individually indicated to be incorporated by referencein their entirety.

In view of the many possible embodiments to which the principles of thedisclosure may be applied, it should be recognized that the illustratedembodiments are only examples and should not be taken as limiting thescope of the invention. Rather, the scope of the invention is defined bythe following claims. We therefore claim as our invention all that comeswithin the scope and spirit of these claims.

TABLE 3 Association of variants on chromosome 22 with FSGS usingFisher's exact test Frequency Frequency derived derived Position Derivedallele in allele in Ancestral Variant (NCBI 36) allele cases controlsallele P-value rs11089781 34886714 A 0.32 0.21 G 0.001341 rs736414334932129 T 0.56 0.4 G 9.904e−06 rs7289037 34938336 A 0.53 0.33 G5.118e−08 rs8136528 34941252 T 0.52 0.34 C 4.977e−07 rs4821469 34946391C 0.7 0.51 T 9.835e−08 rs73885303 34953617 T 0 0 C 0.4759  rs1085468734954365 A 0.51 0.3 C 4.117e−09 rs9622362 34986390 A 0.04 0.09 C0.01488  rs9622363 34986501 G 0.82 0.51 A 6.112e−20 ns41297245 34987686A 0.01 0.05 G 0.000927 rs2239785 34991276 A 0.14 0.35 G 3.081e−12rs136175 34991512 A 0.97 0.93 G 0.01712  rs73403889 34991637 A 0 0 G1     rs16996616 34991837 A 0.03 0.08 G 0.00933  rs73885319 34991852 G0.53 0.19 A  1.07e−23 rs60910145 34991980 G 0.52 0.18 T 2.591e−23rs71785313 34991997 D 0.23 0.15 I 0.008882 rs58384577 34993159 C 0.490.19 T 9.782e−18 rs60295735 34997100 A 0.5 0.2 G 9.128e−17 rs5627760234998706 G 0.5 0.76 T 2.359e−13 rs73885325 35000629 T 0.49 0.25 A6.903e−12 SNP 2 bp after 35005098 A 0.44 0.22 G 1.482e−09 rs136196rs73405714 35005359 G 0.44 0.22 A 9.451e−10 rs11703176 35008422 A 0.480.31 C 3.782e−06 rs58168942 35012680 A 0.4 0.19 G 2.652e−10 rs575613035014277 T 0.06 0.1 C 0.08136  rs6000226 35014513 T 0.32 0.26 C 0.09085 rs11912763 35014668 A 0.41 0.19 G 9.573e−12 rs11549907 35014926 T 0.060.08 C 0.2976  rs6000229 35016105 T 0.2 0.36 C 5.378e−06 rs600022335017908 0 0 0 A 1     rs73405726 35018652 C 0.47 0.29 G  3.09e−07rs73405727 35020433 T 0.42 0.19 C 2.243e−11 rs2239786 35021873 C 0.320.29 G 0.4288  rs16996648 35022698 C 0.47 0.29 T  3.09e−07 rs5633945935023558 T 0.42 0.19 C 8.107e−12 rs4821481 35025888 T 0.17 0.36 C4.398e−09 rs6000235 35026033 T 0.78 0.53 C 7.393e−13 rs3752462 35040129T 0.87 0.72 C  1.82e−07 rs2239784 35044581 T 0.72 0.63 C 0.005961rs8141189 35044656 T 0.57 0.7 A  0.0002506 rs7285770 35045413 A 0.410.25 G 5.869e−06 rs55816447 35047283 T 0.42 0.25 C 7.291e−07 rs5567083035049098 G 0.61 0.76 A 2.948e−05 rs16996668 35049131 G 0.58 0.75 C 2.92e−06 rs12160045 35049306 G 0.55 0.68 A  0.0004368 rs1191213935052407 T 0.41 0.25 C  1.34e−06 rs11912881 35053384 A 0.41 0.25 T8.857e−06 rs16996672 35055916 T 0.45 0.27 C 1.611e−07 rs1699667435056598 T 0.41 0.25 C 3.157e−06 rs16996677 35057229 A 0.45 0.26 G6.587e−08

TABLE 4 Number and frequencies of APOL1 genotypes and alleles in FSGSand hypertension-attributed ESKD cases and controls Hypertension- FSGSCases and Controls attributed ESKD BWH NIH Total NIH WFU WFU Cases CasesCases Controls Cases Controls Genotype WT + WT 3 26 29 77 239 409 WT +G1 6 21 27 41 173 250 WT + G2 0 9 9 36 124 155 G1 + G1 25 35 60 9 219 41G1 + G2 15 38 53 8 203 50 G2 + G2 3 11 14 5 44 18 Total 52 140 192 1761002 923 Allele G1 Freq. 0.68 0.47 0.52 0.18 0.41 0.21 G2 Freq. 0.190.25 0.23 0.15 0.21 0.13Samples for which either the G1 or G2 assay failed are not reported

TABLE 5 Association of different variants on chromosome 22 with FSGSafter controlling for variant rs73885319 (which co- segregates withrs60910145) using logistic regression Position Variant (NCBI 36) P-valuers11089781 34886714 0.925 rs7364143 34932129 0.9919 rs7289037 349383360.2779 rs8136528 34941252 0.5498 rs4821469 34946391 0.05454 rs7388530334953617 0.9993 rs10854687 34954365 0.3387 rs9622362 34986390 0.8758rs9622363 34986501 0.0001509 rs41297245 34987686 0.1298 rs223978534991276 0.00566 rs136175 34991512 0.2976 rs73403889 34991637 0.9993rs16996616 34991837 0.5397 rs73885319 34991852 NA rs60910145 349919800.6051 rs71785313 34991997 4.377e−07 rs58384577 34993159 0.1982rs60295735 34997100 0.6759 rs56277602 34998706 0.3886 ts7388532535000629 0.3589 SNP 2bp after 35005098 0.1207 rs136196 rs7340571435005359 0.1746 rs11703176 35008422 0.02567 rs58168942 35012680 0.2972rs5756130 35014277 0.2318 rs6000226 35014513 0.0001059 rs1191276335014668 0.4741 rs11549907 35014926 0.9798 rs6000229 35016105 0.1074rs6000223 35017908 NA rs73405726 35018652 0.1368 rs73405727 350204330.5361 rs2239786 35021873 0.0005036 rs16996648 35022698 0.1368rs56339459 35023558 0.3864 rs4821481 35025888 0.003736 rs600023535026033 0.002684 rs3752462 35040129 0.003073 rs2239784 35044581 0.743rs8141189 35044656 0.3762 rs7285770 35045413 0.3736 rs55816447 350472830.3485 rs55670830 35049098 0.2393 rs16996668 35049131 0.4054 rs1216004535049306 0.1751 rs11912139 35052407 0.289 rs11912881 35053384 0.3462rs16996672 35055916 0.6268 rs16996674 35056598 0.3884 rs1699667735057229 0.7119

TABLE 6 Association of different variants on chromosome 22 with FSGSafter controlling for variants rs73885319 and rs71785313 using logisticregression Position Variant (NCBI 36) P-value rs11089781 34886714 0.2439rs7364143 34932129 0.1461 rs7289037 34938336 0.8734 rs8136528 349412520.3631 rs4821469 34946391 0.3533 rs73885303 34953617 0.9993 rs1085468734954365 0.4109 rs9622362 34986390 0.13 rs9622363 34986501 0.8217rs41237245 34987686 0.5326 rs2239785 34991276 0.9337 rs136175 349915120.8123 rs73403889 34991637 0.9993 rs16996616 34991837 0.3839 rs7388531934991852 NA rs60910145 34991980 0.7417 rs71785313 34991997 NA rs5838457734993159 0.3419 rs60235735 34997100 0.9987 rs56277602 34998706 0.5788rs73885325 35000629 0.5589 SNP 2bp after 35005098 0.285 rs136196rs73405714 35005359 0.3297 rs11703176 35008422 0.1264 rs5816894235012680 0.4001 rs5756130 35014277 0.5038 rs6000226 35014513 0.1419rs11912763 35014668 0.529 rs11549907 35014926 0.4356 rs6000229 350161050.9112 rs6000223 35017908 NA rs73405726 35018652 0.3053 rs7340572735020433 0.5282 rs2239786 35021873 0.1924 rs16996648 35022698 0.3063rs56339459 35023558 0.4316 rs4821481 35025888 0.2728 rs6000235 350260330.4815 rs3752462 35040129 0.1644 rs2239784 35044581 0.1031 rs814118935044656 0.5344 rs7285770 35045413 0.5092 rs55816447 35047283 0.4334rs55670830 35049098 0.3437 rs16996668 35049131 0.5467 rs1216004535049306 0.45 rs11912139 35052407 0.3368 rs11912881 35053384 0.4146rs16996672 35055916 0.8562 rs16996674 35056598 0.4494 rs1699667735057229 0.954

TABLE 7 Association of variants on chromosome 22 with hypertensive ESKDusing basic association test Frequency Frequency derived derivedPosition Derived allele allele Ancestral Variant (NCBI 36) allele incases in controls allele P-value rs5999985 34452302 A 0.05 0.04 G 0.3545rs41283201 34452326 A 0.07 0.06 T 0.2416 rs2157258 34672336 C 0.31 0.33T 0.2191 rs16996299 34778586 T 0.41 0.34 C 3.215e−06 rs6000152 34868999A 0.08 0.07 G 0.3209 rs7284379 34881360 T 0.28 0.19 C 8.155e−11rs11089781 34886714 A 0.29 0.21 G 1.327e−10 rs132653 34886769 T 0.440.48 G  0.01523 rs6000173 34917169 T 0.74 0.66 G 2.658e−08 rs6173081934917292 T 0.08 0.11 C  0.000395 rs2016708 34948899 T 0.42 0.25 C1.448e−29 rs1001293 34960895 T 0.45 0.31 C 3.059e−20 rs9622363 34986501G 0.72 0.53 A 6.125e−34 rs136168 34990788 A 0.44 0.54 G 2.395e−10rs16996616 34991837 A 0.05 0.08 G  0.000549 rs73885319 34991852 G 0.410.21 A 1.097e−39 rs71785313 34991997 D 0.21 0.13 I 7.276e−10 rs707835007860 G 0.1 0.15 A  3.42e−05 rs12107 35007928 A 0.07 0.11 G 1.731e−05rs16996639 35008348 A 0.1 0.08 G 0.0599 rs11089787 35008399 G 0.48 0.38C 1.642e−10 rs735853 35009159 G 0.07 0.11 C 1.144e−06 rs5816894235012680 A 0.34 0.2 G 7.018e−24 rs5756129 35014038 C 0.15 0.21 T6.297e−07 rs11912763 35014668 A 0.34 0.19 G 8.035e−24 rs5602067635020066 C 0.4 0.26 T 1.324e−19 rs73885341 35021424 A 0.4 0.27 G5.333e−17 rs4821480 35025193 T 0.26 0.4 G 6.622e−21 rs2032487 35025374 T0.25 0.38 C 3.941e−20 rs4821481 35025888 T 0.26 0.4 C 8.434e−21rs2413396 35038030 C 0.72 0.58 T 1.114e−16 rs5750250 35038429 G 0.68 0.5A 2.757e−26 rs3752462 35040129 T 0.81 0.73 C  5.73e−09 rs1191288135053384 A 0.34 0.25 T 5.993e−11 rs16996674 35056598 T 0.34 0.23 C6.825e−13 rs16996677 35057229 A 0.36 0.27 G 1.709e−10

TABLE 8 Association of different variants on chromosome 22 withhypertensive ESKD after controlling for variant rs73885319 usinglogistic regression Position Variant (NCBI 36) P-value rs599998534452302 0.2828 rs41283201 34452326 0.3638 rs2157258 34672336 0.02322rs16996299 34778586 0.0567 rs6000152 34868999 0.2081 rs7284379 348813600.004519 rs11089781 34886714 0.006513 rs132653 34886769 0.5453 rs600017334917169 0.0008435 rs61730819 34917292 0.1326 rs2016708 349488999.595e−06 rs1001293 34960895 0.006298 rs9622363 34986501 6.124e−08rs136168 34990788 0.01352 rs16996616 34991837 0.5301 rs73885319 34991852NA rs71785313 34991997 8.798e−18 rs7078 35007860 0.1648 rs12107 350079280.05038 rs16996639 35008348 0.002146 rs11089787 35008399 0.156 rs73585335009159 0.0159 rs58168942 35012680 0.9944 rs5756129 35014038 0.06101rs11912763 35014668 0.3702 rs56020676 35020066 0.5497 rs7388534135021424 0.9711 rs4821480 35025193 6.763e−06 rs2032487 350253742.137e−05 rs4821481 35025888 8.516e−06 rs2413396 35038030 0.0006269rs5750250 35038429 4.145e−08 rs3752462 35040129 0.02872 rs1191288135053384 0.8152 rs16996674 35056598 0.47 rs16996677 35057229 0.9985

TABLE 9 Frequency differentiation analysis of variants near APOL1 fortwo African populations, Yoruba from Nigeria and Luhya from KenyaFrequency Frequency Non reference non- Position Reference referenceallele in reference Variant (NCBI 36) allele allele YRI allel in LWKF_(ST) P-value rs12185880 34900774 C G 0.86 0.76 0.02 0.0453 rs13268134904713 A G 0.07 0.05 0 0.5108 rs132683 34905610 G A 0.67 0.58 0.010.1986 rs132686 34906596 A G 1 0.97 0.02 0.0815 rs132688 34906886 G A 10.97 0.01 0.1726 rs6000164 34907077 C T 0.89 0.8 0.02 0.0869 rs13268934907092 G A 0.9 0.95 0.01 0.1459 rs5995235 34907888 C T 0.67 0.62 0.010.3792 rs6000167 34908800 G A 0.45 0.52 0.01 0.3370 rs132692 34909112 TC 0.03 0.16 0.05 0.0012 rs132693 34909507 A G 0.01 0.02 0 0.6093rs2239831 34913030 T C 0.13 0.21 0.01 0.1274 rs916338 34914376 T C 0.010.02 0 0.6093 rs132697 34914659 A G 0.01 0.07 0.02 0.0319 rs813606434914892 T G 0.98 0.91 0.03 0.0201 rs1053982 34915510 T C 0.25 0.41 0.030.0133 rs5756091 34915667 T G 0.24 0.41 0.03 0.0087 rs5756093 34915917 GA 1 1 NaN NaN rs6000172 34917148 G A 0.24 0.37 0.02 0.0413 rs600017434917225 A G 0.24 0.37 0.02 0.0407 rs2227167 34917432 A G 0.24 0.37 0.020.0378 rs2269596 34920892 C T 0.23 0.3 0.01 0.2156 rs2007468 34921326 AG 0.12 0.14 0 0.6150 rs2007706 34922316 C T 0.01 0.1 0.04 0.0059rs132717 34926598 C T 0.23 0.44 0.05 0.0015 rs132734 34927823 G A 0.230.43 0.05 0.0012 rs132735 34927827 G T 0.52 0.67 0.02 0.0307 rs599525134930704 A T 0.61 0.36 0.06 0.0003 rs6000190 34930787 A G 0.61 0.36 0.060.0003 rs5995252 34931145 C T 0.6 0.36 0.06 0.0004 rs7364143 34932129 GT 0.53 0.79 0.08 5.315e−05 rs5995255 34932725 G T 0.58 0.38 0.04 0.0038rs6000197 34933240 G A 0.58 0.37 0.04 0.0029 rs132744 34934551 T C 0.480.27 0.05 0.0015 rs132745 34935277 C T 0.15 0.32 0.04 0.0040 rs13274634935337 C T 0.15 0.32 0.04 0.0030 rs8142325 34935923 A T 0.54 0.83 0.098.998e−06 rs132749 34936575 C T 0.81 0.67 0.03 0.0262 rs9610448 34938151A G 0.22 0.31 0.01 0.1381 rs132750 34938295 C T 0.03 0.04 0 0.4781rs7289037 34938336 G A 0.51 0.81 0.1 6.039e−06 rs11704479 34939580 G A 11 NaN NaN rs4820222 34939685 C T 0.22 0.32 0.02 0.0975 rs600019934939878 G A 0.92 0.84 0.02 0.0661 rs8140384 34940517 C T 0.21 0.22 00.7640 rs8136528 34941252 C T 0.52 0.79 0.09 1.990e−05 rs599525934941809 G A 0.82 0.76 0.01 0.3005 rs1315 34946081 A C 0.9 0.9 0 0.9650rs4821467 34946146 G A 0.51 0.79 0.09 1.390e−05 rs4821469 34946391 T C0.34 0.45 0.02 0.0877 rs763086 34949003 G A 0.34 0.49 0.03 0.0273rs11703398 34950907 A G 0.83 0.7 0.03 0.0223 rs2006259 34951559 A C 0.350.49 0.02 0.0366 rs132757 34951655 T C 0 0.01 0.01 0.4369 rs961959734952768 G T 1 1 NaN NaN rs129607 34952852 T C 0.39 0.64 0.06 0.0003rs132760 34953677 T C 0 0 NaN NaN rs7285167 34953866 G A 0.54 0.86 0.123.653e−07 rs11089784 34956223 C T 0.9 0.9 0 0.8770 rs11703957 34956901 AG 0.79 0.72 0.01 0.2647 rs2010467 34958853 T C 0.54 0.24 0.1 6.779e−06rs2010659 34959579 A C 0.86 0.87 0 0.8853 rs9610462 34960296 C A 0.860.87 0 0.8162 rs1001294 34960936 C T 0.86 0.86 0 0.9329 rs215724934960985 T C 0.86 0.86 0 0.9545 rs2157250 34961637 G A 0.05 0.03 00.4757 rs136142 34962971 C T 0.65 0.44 0.05 0.0024 rs1557534 34963171 GA 0.97 0.94 0.01 0.3752 rs136145 34965913 A G 0.3 0.48 0.04 0.0063rs4821472 34977906 T C 0.97 0.91 0.02 0.0473 rs5995271 34978039 G T 0.930.84 0.02 0.0496 rs5756115 34978498 A G 1 0.96 0.02 0.0804 rs961046734979520 G A 0.91 0.84 0.01 0.1329 rs7284919 34982110 T C 0.93 0.83 0.030.0229 rs136148 34982877 C T 0.1 0.16 0.01 0.1986 rs4820224 34983221 G A0.99 0.97 0.01 0.4534 rs2413395 34984662 G A 0.93 0.92 0 0.7441 rs13615934986969 T C 0 0.04 0.02 0.0390 rs129423 34987275 T C 0 0.03 0.02 0.0551rs136161 34987378 G C 0.8 0.65 0.03 0.0143 rs713929 34987542 A G 0 0.030.02 0.0561 rs713753 34988480 C T 0.89 0.72 0.04 0.0026 rs441933034988801 T C 0.89 0.89 0 0.8258 rs2239785 34991276 G A 0.73 0.56 0.030.0088 rs136174 34991432 C A 0 0.07 0.03 0.0137 rs136175 34991512 G A 00.07 0.03 0.0137 rs136176 34991592 G A 0 0.04 0.03 0.0270 rs13617734991788 G A 0.03 0.13 0.04 0.0063 rs16996616 34991837 G A 0.94 0.870.02 0.0974 rs73885319 34991852 G A 0.38 0.05 0.16 3.533e−09 rs7178531334991997 D I 0.08 0.07 0 0.8949 rs2012928 34993948 G A 0.83 0.67 0.030.0086 rs136183 34996271 T C 0.34 0.55 0.05 0.0022 rs4821475 34999041 CT 0.37 0.46 0.01 0.1825 rs9306308 34999716 T A 0.98 0.86 0.05 0.0015rs136187 35002222 A C 0.5 0.62 0.02 0.0783 rs136196 35005096 A G 0.310.37 0.01 0.4163 rs2481 35007346 G A 0.91 0.77 0.04 0.0063 rs73585435009004 T C 0.93 0.78 0.05 0.0014 rs5756129 35014038 T C 0.79 0.7 0.010.1210 rs5756130 35014277 C T 0.82 0.83 0 0.8596 rs2269529 35014300 T C0.97 0.87 0.04 0.0039 rs2269530 35014304 C A 0.97 0.88 0.03 0.0094rs11912763 35014668 G A 0.67 0.94 0.12 4.030e−07 rs1476009 35016002 A G0.04 0.02 0 0.5721 rs6000229 35016105 T C 0.28 0.34 0.01 0.3473rs6000233 35017908 T C 0.64 0.47 0.03 0.0098 rs710181 35021553 A C 0.020.02 0 0.9960 rs75725 35021637 T C 0.93 0.94 0 0.6467 rs2239786 35021873G C 0.71 0.53 0.03 0.0085 rs875726 35021915 G A 0.81 0.82 0 0.8952rs16996648 35022698 T C 0.6 0.88 0.1 3.222e−06 rs9610486 35023388 G A0.81 0.82 0 0.7884 rs5756133 35023926 T A 0.91 0.95 0.01 0.2300rs2187776 35025119 C T 0.31 0.46 0.02 0.0276 rs4821481 35025888 C T 0.730.64 0.01 0.1505 rs2239787 35028938 C A 1 0.99 0 0.9095 rs961960135030121 A G 0.96 0.97 0 0.9326 rs8137674 35032048 A G 0.99 0.94 0.020.0888 rs8138016 35032095 G A 0.94 0.96 0 0.5345 rs17806543 35034780 C A1 0.99 0.01 0.4376 rs2239781 35034987 C T 0.98 0.89 0.04 0.0052rs2239782 35035050 G A 0.95 0.87 0.02 0.0403 rs1557529 35035475 A G 0.520.39 0.02 0.0577 rs1557530 35035568 G A 0.79 0.8 0 0.8724 rs218777735036688 C T 1 0.99 0.01 0.2717 rs2157252 35036825 C A 0.77 0.79 00.7517 rs2157254 35037146 G C 0.77 0.79 0 0.7517 rs2157256 35037607 A G0.71 0.69 0 0.7725 rs2413396 35038030 C T 0.67 0.54 0.02 0.0633rs5750250 35038429 G A 0.64 0.54 0.01 0.1494 rs3830104 35038570 T C 0.970.99 0.01 0.2224 rs4820229 35038699 A G 0.77 0.79 0 0.7517 rs482023035039485 G A 0.71 0.7 0 0.8089 rs3752462 35040129 T C 0.77 0.79 0 0.7954rs4820232 35040487 A G 0.75 0.75 0 0.9919 rs8141971 35041308 A G 0.780.79 0 0.8734 rs5756152 35042418 A G 0.42 0.33 0.01 0.2106 rs961048935043477 T C 0.91 0.73 0.05 0.0010 rs2239784 35044581 C T 0.3 0.48 0.040.0050 rs1005570 35045220 A G 0.5 0.43 0.01 0.2882 rs2071731 35048804 GA 0.79 0.74 0.01 0.4006 rs12159211 35049109 G A 0.99 0.99 0 0.5521rs5756154 35050370 C T 0.7 0.72 0 0.7700 rs5756156 35050725 C T 0.750.77 0 0.6751 rs8136069 35052436 C A 0.79 0.74 0.01 0.4006 rs813633635052480 G A 0.04 0.02 0.01 0.3053 rs16996672 35055916 C T 0.63 0.780.03 0.0151 rs16996677 35057229 G A 0.63 0.79 0.03 0.0113 rs1170438235058098 C A 1 1 NaN NaN ns4820234 35059020 A G 0.27 0.29 0 0.7013rs2413398 35060893 T G 0.73 0.74 0 0.8116 rs1557540 35062483 C T 0.260.28 0 0.7824 rs713839 35063884 A G 0.73 0.74 0 0.8269 rs739096 35071686G C 0.97 0.94 0.01 0.2819 rs6000244 35071832 C T 0.94 0.85 0.02 0.0357rs739097 35076025 G A 0.35 0.36 0 0.9082 rs5756164 35078939 A G 0.050.09 0.01 0.2460 rs11089788 35081047 C A 0.56 0.63 0.01 0.3345 rs13620635085444 A G 0.67 0.61 0.01 0.3496 rs16996693 35086202 A C 0.99 0.99 00.7813 rs9306310 35088204 G A 0.99 0.94 0.02 0.0367 rs136211 35088493 AG 0.45 0.38 0.01 0.3437 rs16996704 35094734 A G 0.46 0.52 0.01 0.3706rs933224 35095949 T C 0.42 0.41 0 0.9430 rs1883273 35099631 G A 0.590.54 0 0.5139 rs6000262 35099984 A G 0.59 0.55 0 0.5608

TABLE 10 Subject Characteristics (n = 407) Mean ± SD Range Percent (n)(Min-Max) Age at Dialysis Initiation 55.2 ± 17.1 18.9-94.7  Sex Male52.3% (213) Female 47.7% (194) Median Income Tertile Three 31.9% (130)$31,924-$107,479 Tertile Two 33.2% (135) $21,076-$31,611  Tertile One32.4% (132) $6,878-$20,985 Unknown 2.5% (10) Census Region Northeast11.3% (46) Midwest/West 17.0% (69) South 69.8% (284) Unknown 2.0% (8)Access Catheter 58.5% (238) Graft 11.1% (45) Fistula 23.3% (95) Unknown7.1% (29) Location of Dialysis Initiation Inpatient 83.5% (340)Outpatient 16.5% (67) Cause of ESRD Hypertension 72.7% (296) Other 27.0%(110) Unknown 0.3% (1) Body Mass Index 27.2 ± 7.8  13.8-67.5  SystolicBlood Pressure, mm Hg 145.0 ± 22.4  90.0-219.0 Diastolic Blood Pressure,mm Hg 79.4 ± 14.1 49.0-137.0 Albumin, g/dl 3.4 ± 0.6 1.3-4.7 Creatinine, mg/dl 8.1 ± 3.6 2.1-22.1 eGFR, mL/min/1.73 m² 9.9 ± 5.63.0-45.4 PTH, pg/ml 387.9 ± 325.8   4.6-2,353.4 Calcium, mg/dl 8.4 ± 1.04.3-12.7 Hemoglobin, g/dl 9.9 ± 1.3 5.9-14.9 eGFR = Estimated GlomerularFiltration Rate

TABLE 11 Subject Characteristics by G1 Risk Allele Status Wild TypeHeterozygous Homozygous (n = 104) (n = 101) (n = 85) p-value Age atDialysis Initiation*   61.8 ± 17.1^(a, b)  55.9 ± 16.7^(a)  49.0 ±14.9^(b) 1.0 × 10⁻⁶ Sex 0.9314 Male 51.0% (53) 53.5% (54) 52.9% (45)Female 49.0% (51) 46.5% (47) 47.1% (40) Median Income 0.6869 Thirdtertile 26.9% (28) 34.7% (35) 35.3% (30) Second fertile 29.8% (31) 33.7%(34) 32.9% (28) First tertile 37.5% (39) 30.7% (31) 31.8% (27) Unknown5.8% (6) 1.0% (1) 0.0% (0) Census Region 0.7810 Northeast 11.5% (12)13.9% (14) 9.4% (8) Midwest/West 20.2% (21) 15.8% (16) 17.7% (15) South64.4% (67) 69.3% (70) 72.9% (62) Unknown 3.9% (4) 1.0% (1) 0.0% (0)Access 0.8344 Catheter 57.7% (60) 63.4% (64) 58.8% (50) Graft 15.4% (16)10.9% (11) 12.9% (11) Fistula 19.2% (20) 20.8% (21) 23.5% (20) Unknown7.7% (8) 5.0% (5) 4.7% (4) Location of Dialysis Initiation 0.4177Inpatient 78.9% (82) 85.2% (86) 84.7% (72) Outpatient 21.2% (22) 14.9%(15) 15.3% (13) Cause of ESRD 0.9573 Hypertension 73.1% (76) 71.3% (72)71.8% (61) Other 26.9% (28) 28.7% (29) 27.1% (23) Unknown 0.0% (0) 0.0%(0) 1.2% (1) Body Mass Index 25.9 ± 6.3  26.9 ± 7.5  27.6 ± 7.9  0.2838Systolic Blood Pressure, mm Hg 146.6 ± 22.3  145.4 ± 23.5  141.3 ± 20.6 0.2514 Diastolic Blood Pressure, mm Hg 77.8 ± 12.9 79.3 ± 15.2 80.6 ±14.7 0.3898 Albumin, g/dl 3.5 ± 0.6 3.4 ± 0.6 3.5 ± 0.6 0.6550Creatinine, mg/dl*   6.8 ± 2.8^(a, b)  7.7 ± 3.4^(a)  9.4 ± 3.8^(b) 1.0× 10⁻⁶ eGFR, mL/min/1.73 m²   11.6 ± 6.6^(a, b) 10.4 ± 5.5^(a)  8.0 ±3.5^(b) 8.1 × 10⁻⁵ PTH, pg/ml 332.2 ± 318.7 3337.2 ± 249.7  444.9 ±382.1 0.0585 Calcium, mg/dl 8.5 ± 0.9 8.4 ± 1.0 8.3 ± 1.0 0.5680Hemoglobin, g/dl 9.9 ± 1.3 9.8 ± 1.4 10.1 ± 1.2  0.3316 *Values with thesame letter differ significantly from each other based on post-hoc testseGFR = Estimated Glomerular Filtration Rate

TABLE 12 Average Predicted Age at Dialysis Initiation from LinearRegression Models by G1 Risk Allele Wild Type Heterozygous^(c)Homozygous^(c) Age 61.8 55.9 49.0 — p = 0.011 p = 2.1 × 10⁻⁷ Age +Hypertensive ESRD 61.8 55.9 49.1 — p = 0.011 p = 1.710⁻⁷   Age +Hypertensive ESRD + Male Sex 61.8 55.9 49.1 — p = 0.012 p = 1.710⁻⁷  Age + Hypertensive ESRD + Male Sex + Third 62.1 55.8 49.1 TertileIncome^(a) + First Tertile Income^(a) —    p = 8.0 × 10⁻³ p = 1.210⁻⁷  Age + Hypertensive ESRD + Male Sex + Third 62.1 55.8 49.1 TertileIncome^(a) + First Tertile Income^(a) + — p = 0.012 p = 2.1 × 10⁻⁷Inpatient Dialysis Age + Hypertensive ESRD + Male Sex + Third 62.1 55.949.1 Tertile Income^(a) + First tertile Income^(a) + — p = 0.013 p = 1.7× 10⁻⁷ Inpatient Dialysis + Northeast Region^(b) + South Region^(b)P-values represent significance of G1 risk allele coefficientscontrolling for all other variables in the model ^(a)Reference group isMedium Income ^(b)Reference group is Midwest/West ^(c)Reference group isWild Type

1-88. (canceled)
 89. A method for selecting a human subject fortreatment to reduce the risk of developing renal failure or delay thedevelopment of renal failure, the method comprising: determining thepresence of at least one apolipoprotein L1 (APOL1) gene risk allele inthe subject.
 90. The method of claim 89, wherein the at least one humanAPOL1 gene risk allele comprises at least one single nucleotidepolymorphism (SNP) and/or at least one inversion in a human APOL1 gene.91. The method of claim 90, wherein the at least one SNP produces anAPOL1 polypeptide having a serine to glycine mutation at position 342(S342G), an isoleucine to methionine mutation at position 384 (I384M), adeletion of amino acids N388 and Y389, or a combination thereof.
 92. Themethod of claim 91, wherein the at least one SNP produces an APOL1polypeptide having a S342G and an I384M mutation.
 93. The method ofclaim 90, wherein the method comprises determining the presence of theat least one SNP and/or the at least one inversion on both chromosomesof the subject.
 94. The method of claim 89, wherein the renal disease isfocal segmental glomerulosclerosis (FSGS) or hypertensive end-stagekidney disease, or both.
 95. The method of claim 89, wherein the subjectis of African or Hispanic ancestry.
 96. The method of claim 90,comprising determining the presence of the at least SNP and the at leastone inversion in the human APOL1 gene.
 97. The method of claim 90,wherein the inversion comprises recombination between said human APOL1gene and a human apolipoprotein 4 (APOL4) gene or the inversioncomprises substitution of the 5′ region of said APOL1 gene with the 5′region of an APOL4 gene
 98. The method of claim 90, wherein saidinversion occurs in a coding region of said APOL1 gene, a non-codingregion of said APOL1 gene, or in both regions.
 99. The method of claim89, wherein the nucleic acid molecule having the sequence of at leastone APOL1 gene risk allele or a complement thereof has a nucleotidesequence of SEQ ID NO:1, SEQ ID NO:2 and/or SEQ ID NO:3 or a complementthereof, or the nucleic acid primer capable of amplifying the nucleicacid molecule or complement thereof has a nucleotide sequence of SEQ IDNO:1, SEQ ID NO:2 and/or SEQ ID NO:3.
 100. The method of claim 99,wherein the determining further comprises determining the presence orabsence of a G allele at single nucleotide polymorphism (SNP) rs73885319in the nucleotide sequence of SEQ ID NO:1, the presence or absence of aG allele at SNP rs60910145 in the nucleotide sequence of SEQ ID NO:2,and the presence or absence of a six base pair deletion at SNPrs71785313 in the nucleotide sequence of SEQ ID NO:3.
 101. The method ofclaim 89, wherein the biological sample is selected from the groupconsisting or whole blood, serum, buccal cells, extracted galls,biopsied or surgically removed tissue, tears, milk, a skin scrape, asurface washing, urine, sputum, cerebrospinal fluid, prostate fluid,pus, and a bone marrow aspirate.
 102. The method of claim 89, whereintesting the subject for the presence of at least one APOL1 gene riskallele comprises: i) contacting a biological sample from the humansubject with a nucleic acid probe capable of hybridizing to a nucleicacid molecule having the sequence of at least one apolipoprotein L1(APOL1) gene risk allele or a complement thereof, or a nucleic acidprimer capable of amplifying the nucleic acid molecule or complementthereof; ii) detecting formation of a hybridization complex between thenucleic acid probe and the nucleic acid molecule or complement thereofor an amplification product corresponding to the nucleic acid moleculeor complement thereof; and iii) selecting the human subject fortreatment to reduce the risk of developing renal failure renal failureor delay the development of renal failure in which the formation of ahybridization complex or an amplification product is detected.
 103. Amethod for selecting a human subject in need of screening for renaldisease and/or for the risk of developing renal disease, the methodcomprising: determining the presence of an apolipoprotein L1 (APOL1)gene risk allele in a biological sample of the subject; and selectingthe subject in which the APOL1 gene risk allele is present in thebiological sample for screening for renal disease and/or for the risk ofdeveloping renal injury/disease.
 104. The method of claim 103, whereinthe screening of the subject is an annual or biannual screening forrenal disease and/or for risk of developing renal injury/disease. 105.The method of claim 103, wherein the APOL1 gene risk allele comprises atleast one SNP and/or at least one inversion in a human APOL1 gene. 106.The method of claim 105, wherein the at least one SNP produces an APOL1polypeptide having a serine to glycine mutation at position 342 (S342G),an isoleucine to methionine mutation at position 384 (I384M), a deletionof amino acids N388 and Y389, or a combination thereof.
 107. The methodof claim 106, wherein the at least one SNP produces an APOL1 polypeptidehaving a S342G and an I384M mutation.
 108. The method of claim 105,wherein the method comprises determining the presence of the at leastone SNP and/or the at least one inversion on both chromosomes of thesubject.
 109. The method of claim 103, wherein the renal disease isfocal segmental glomerulosclerosis (FSGS) or hypertensive end-stagekidney disease, or both.
 110. The method of claim 103, wherein thesubject is of African or Hispanic ancestry.
 111. The method of claim105, comprising determining the presence of the at least SNP and the atleast one inversion in the human APOL1 gene.
 112. The method of claim105, wherein the inversion occurs in a coding region of said APOL1 gene,a non-coding region of said APOL1 gene, or in both regions.
 113. Themethod of claim 105, wherein the inversion comprises recombinationbetween said human APOL1 gene and a human apolipoprotein 4 (APOL4) geneor the inversion comprises substitution of the 5′ region of said APOL1gene with the 5′ region of an APOL4 gene.
 114. The method of claim 103,wherein the at least one APOL1 gene risk allele comprises a nucleotidesequence of SEQ ID NO:1, SEQ ID NO:2 and/or SEQ ID NO:3 or a complementthereof.
 115. The method of claim 114, wherein the determining furthercomprises determining the presence or absence of a G allele at singlenucleotide polymorphism (SNP) rs73885319 in the nucleotide sequence ofSEQ ID NO:1, the presence or absence of a G allele at SNP rs60910145 inthe nucleotide sequence of SEQ ID NO:2, and the presence or absence of asix base pair deletion at SNP rs71785313 in the nucleotide sequence ofSEQ ID NO:3.
 116. The method of claim 103, wherein the determiningfurther comprises a) contacting the biological sample from the humansubject with a nucleic acid probe capable of hybridizing to a nucleicacid molecule having the sequence of at least one apolipoprotein L1(APOL1) gene risk allele or a complement thereof, or a nucleic acidprimer capable of amplifying the nucleic acid molecule or complementthereof; b) determining formation of a hybridization complex between thenucleic acid probe and the nucleic acid molecule or complement thereofor an amplification product corresponding to the nucleic acid moleculeor complement thereof; and c) selecting the human subject for screeningfor renal disease and/or risk of developing renal disease in which theformation of a hybridization complex or an amplification product isdetected.
 117. The method of claim 116, wherein the nucleic acidmolecule having the sequence of at least one APOL1 gene risk allele or acomplement thereof has a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2and/or SEQ ID NO:3 or a complement thereof, or the nucleic acid primercapable of amplifying the nucleic acid molecule or complement thereofhas a nucleotide sequence of SEQ ID NO:1, SEQ ID NO:2 and/or SEQ IDNO:3.
 118. The method of claim 117, wherein the determining furthercomprises determining the presence or absence of a G allele at singlenucleotide polymorphism (SNP) rs73885319 in the nucleotide sequence ofSEQ ID NO:1, the presence or absence of a G allele at SNP rs60910145 inthe nucleotide sequence of SEQ ID NO:2, and the presence or absence of asix base pair deletion at SNP rs71785313 in the nucleotide sequence ofSEQ ID NO:3
 119. The method of claim 103, wherein the biological sampleis selected from the group consisting or whole blood, serum, buccalcells, extracted galls, biopsied or surgically removed tissue, tears,milk, a skin scrape, a surface washing, urine, sputum, cerebrospinalfluid, prostate fluid, pus, and a bone marrow aspirate.